Methods of using il-31 to treat chronic obstructive pulmonary disease (copd)

ABSTRACT

Use of IL-31 agonists, including IL-31, are used to treat agonists are used to treat asthma, airway hyper-responsiveness or allergic rhinitis. The method comprise inhibiting, reducing, limiting or minimizing production of proinflammatory cytokines and include administration of the IL-31 agonist during sensitization or challenge resulting in the asthma, airway hyper-responsiveness or allergic rhinitis state.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/869,421, filed Aug. 26, 2010, which is a divisional of U.S.application Ser. No. 11/972,596, filed Jan. 10, 2008, now U.S. Pat. No.7,799,323, and claims the benefit of U.S. Provisional Application Ser.No. 60/884,379, filed Jan. 10, 2007, all of which are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

Asthma is a chronic lung disease that affects more than 17 millionAmericans. Asthma is characterized by inflammation of the airways withintermittent bronchospasm., which is caused by the inflammation of themuscles surrounding the air passageways. Breathing may be so laboredthat an asthma attack becomes life-threatening. Asthma is a chronicdisease and it requires continuous management and appropriate treatment.

Symptoms of asthma include cough, chest tightness, shortness of breath,and wheezing. Asthma can be triggered by a variety of irritations, suchas allergens, tobacco smoke, strong odors, respiratory infections,weather changes, viral or sinus infections, exercise, stress, refluxdisease (Stomach acid flowing back up the esophagus, or food pipe),medications, foods, and emotional anxiety.

Different classifications of asthma include: allergic asthma, caused byairway inflammation when exposed to allergens; exercised-induced asthma,where the airways narrow when triggered by vigorous activity;cough-variant asthma, a chronic, persistent cough without shortness ofbreath; and occupational asthma, which is related to working in aparticular occupational environment.

Management of asthma involves several approaches, including preventingchronic and troublesome symptoms; maintaining “normal” breathing;maintain normal activity levels, including exercise; preventingrecurrent asthma flare-ups, and minimize the need for emergency roomvisits or hospitalizations, and providing optimal medication therapywith no or minimal adverse effects. Asthma management includes usingproper medications, or combinations of medications to prevent andcontrol asthma symptoms and to reduce airway inflammation. Asthmamedications are thus categorized into two general classes, quick-reliefand long-term control medications. Quick-relief medications that areused to provide temporary relief of symptoms include bronchodilators,such as beta-agonists and anticholinergics, and corticosteroids.Long-term control medications are taken daily to control the airwayinflammation in persistent asthma. This class includes inhaledcorticosteroids to inhibit or prevent inflammation.

Thus, there is a need for additional treatment options in managingasthma and airway hyper-responsiveness. The present invention providesthe use of a cytokine to aid in management of this disease.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 a shows quantitative RT-PCR analysis of IL-31RA and OSMR levelsin lung tissue following sensitization with OVA and challenge witheither OVA or PBS in BALB/c or C57B1/6 mice.

FIG. 1 b shows quantitative RT-PCR analysis of IL-31RA and OSMR levelsin cells from bronchial alveolar lavage fluid (BAL) followingsensitization with OVA and challenge with either OVA or PBS in BALB/c orC57B1/6 mice.

DESCRIPTION OF THE INVENTION

The present invention is based in part upon the discovery that mice in amodel of airway hyper-responsiveness (AHR) that were treated with IL-31indicated exhibited less AHR compared to vehicle treated controls andthat IL-31 treatment decreases disease pathogenesis in a murine model ofallergic asthma, possibly through the down-regulation of IL-5 and IL-13.In addition, the invention teaches the unexpected findings that timingof the administration of IL-31 and dosage are important in using IL-31to treat asthma and ARR. Thus the present invention encompasses the useof IL-31 to treat asthma, acute respiratory distress, chronicobstructive pulmonary disease, allergic rhinitis, and respiratorydiseases.

IL-31 is the HUGO name for a cytokine that has been previously describedas Zcyto17rlig in a published U.S. patent application (See publishedU.S. patent application number 20030224487, U.S. patent application Ser.No. 10/352,554, filed Jan. 21, 2003, now issued U.S. Pat. No. 7,064,186;Sprecher, Cindy et al., 2003, incorporated herein by reference). Theheterodimeric receptor for IL-31 comprises a heterodimer formed betweenIL-31 Ra and OncostatinM receptor beta (OSMRb). IL-31Ra is the HUGO namefor a protein called zcytor17 in commonly-owned U.S. published patentapplication number 20030215838, U.S. patent application Ser. No.10/351,157, filed Jan. 21, 2003, herein incorporated by reference. Thepolynucleotide and polypeptide sequences for human IL-31 are shown inSEQ ID NOs: 1 and 2, respectively. The polynucleotide and polypeptidesequences for murine IL-31 are shown in SEQ ID NOs: 3 and 4,respectively. As used herein the term, IL-31 shall mean zcytor17 lig asused in U.S. patent publication number 20030224487, as shown above.IL-31Ra has been previously described in commonly-owned U.S. patentapplication Ser. No. 09/892,949 filed Jun. 26, 2001, which is hereinincorporated by reference.

Cysteine mutanst of IL-31 are described in U.S. Patent Publication2006-0228329, published Oct. 12, 2006 and are also incorporated hereinby reference. Molecules of the mature human IL-31 polypeptide can havedisulfide bonds between the cysteine residues of the mature polypeptideamino acid sequence A mutation of any of these three cysteines resultsin a mutant form of the human IL-31 protein that will only form onedisulfide bond. The cysteines in these positions can be mutated, forexample, to a serine, alanine, threonine, valine, or asparagine.

The amino acid sequence for the OSMR, and IL-31RA receptors indicatedthat the encoded receptors belonged to the Class I cytokine receptorsubfamily that includes, but is not limited to, the receptors for IL-2,IL-4, IL-7, Lif, IL-15, EPO, TPO, GM-CSF and G-CSF (for a review see,Cosman, “The Hematopoietin Receptor Superfamily” in Cytokine 5(2):95-106, 1993). The zcytor17 receptor is fully described incommonly-owned PCT Patent Application No. US01/20484 (WIPO publicationNo. WO 02/00721; herein incorporated by reference).

The present invention includes the use of IL-31 molecules, includingagonists, variants and fragments, having IL-31 activity to treat asthmaand/or AHR. The invention includes administering to a subject the IL-31molecule and contemplates both human and veterinary therapeutic uses.Illustrative veterinary subjects include mammalian subjects, such asfarm animals and domestic animals.

The native polynucleotide and polypeptide sequences for the “long” formof IL-31RA are shown in SEQ ID NOs: 5 and 6, respectively. The nativepolynucleotide and polypeptide sequences for the “short” form of IL-31RAare shown in SEQ ID NOs: 7 and 8, respectively. Additional truncatedforms of IL-31RA polypeptide appear to be naturally expressed. Bothforms encode soluble IL-31RA receptors. The “long” soluble IL-31RApolynucleotide and polypeptide sequences are shown in SEQ ID NOs: 9 and10, respectively. The “short” soluble IL-31RA polynucleotide andpolypeptide sequences are shown in SEQ ID NOs: 11 and 12, respectively.The native polynucleotide and polypeptide sequences for mouse IL-31RAare shown in SEQ ID NOs: 13 and 14, respectively. The nativepolynucleotide and polypeptide sequences for human OSMRbeta are shown inSEQ ID NOs: 15 and 16, respectively. See PCT applications WO 02/00721and WO 04/003140, both of which are incorporated by reference.

In allergic asthma, inhalation of allergens leads to an inflammatorycascade in which CD4+ T lymphocytes are thought to play a central role.The key contributions of CD4+ T cells in the pathogenesis of asthma havebeen highlighted by studies of Th2-type cytokines, such as IL-4, IL5,IL-9 and IL-13. These cytokines can mediate upregulation of adhesionmolecules and inflammatory chemokine production, and thereby immune-cellrecruitment, degranulation of eosinophils, synthesis of IgE, andhyper-reactivity of smooth muscle (reviewed 1). IL-4 and IL-13 arestructurally related molecules that share the common IL-4Rα chain inreceptor complexes. See Lin J. et al., Immunity 2:331-9, 1995;Smerz-Bertling C., and Duschl A., J. Biol. Chem. 270:966-70, 1995; andZurawski S. et al., J. Biol. Chem. 270:13869-78, 1995. Although theyexhibit overlapping function and both are associated with allergicdisease, studies in IL-4 deficient animals have demonstrated that IL-13may be especially critical for the induction of ABR. See Grunig G. etal., Science 282:2261-3, 1998; Herrick C. et al., J. Immunol.170:2488-95, 2003; and Wills-Karp M. et al., Science 282:2258-61, 1998.IL-5 is central to eosinophil maturation, differentiation, activationand survival. The development of airway eosinophilia is associated withincreased IL-5 expression in the airway mucosa and elevatedconcentrations of IL-5 in the luminal fluid and serum (Liu L. et al., J.Allergy Clin. Immunol.:106:1063-9, 2000; and Kelly E. et al., Am. J.Respir. Crit. Care Med. 156:1421-8, 1997). Additionally, studies in micehave indicated the role of IL-5 in eosinophilia through depletion inmurine models of asthma (Saito H. et al., J. Immunol. 168:3017-23, 2002;Tanaka H. et al., Am. J. Respir. Cell Mol. Biol. 19:19, 2004; and TomakiM. et al., Pulm. Pharmacol. Ther. 15:161-8, 2002). Therefore Th2mediated cytokines play an important role in generating the inflammationthat characterizes allergic diseases.

IL-31 has been found to be produced more predominantly by activated Th2cells compared to Th1-skewed cells (Dillon et al., 2004). Subsequentanalysis of lung tissue from mice exposed to a model of allergen-inducedasthma showed an upregulation of the receptor for IL-31, IL-31RA,suggesting a possible association of IL-31 with allergy. In that study,RNA was isolated from human IL-31 treated A549 cells, IL-31 treatedSK-LU-1 cells, and untreated control cells using a RNeasy Midi Kit(Qiagen, Valencia, Calif.) according to the manufactures instructions.Gene expression profiling of the cells treated with IL-31 and therespective control cells was carried out using GEArray Q series cDNAexpression arrays (SuperArray Inc., Bethesda, Md.). The Q Series cDNAexpression arrays contain up to 96 cDNA fragments associated with aspecific biological pathway, or genes with similar functions orstructural features. Comparison of arrays from treated and control cellsallows for a determination of the up and down regulation of specificgenes. Probe labeling, hybridization and detection were carried outaccording to the manufactures instructions. Chemiluminscent signaldetection and data acquisition was carried out on a Lumi-Imagerworkstation (Roche, Indianapolis, Ind.). The resulting image data wasanalyzed using ImageQuant 5.2 (Amersham Biosciences, Inc., Piscataway,N.J.) and GEArray Analyzer 1.2 (SuperArray Inc., Bethesda, Md.)software. Analysis of the results from the Human Interleukin andReceptor Q series HS-014N arrays, showed, after normalization, anapproximate 4.7 fold increase of IL13RA2 signal in the IL-31 treatedhuman SK-LU-1 cells and an approximate 2.2 fold increase of the IL13RA2signal in the IL-31 treated human A549 cells. These results indicatethat IL-31 significantly up regulated IL13RA2 in the SK-LU-1 and A549cells. Both of these are established cell lines derived from human lungcarcinomas (Blobel et al., Virchows Arch B Cell Pathol Incl Mol.Pathol., 1984; 45(4):407-29). More specifically, A549 is characterizedas a human pulmonary epithelial cell line (Lin, et al., J PharmPharmacol., 2002 September; 54(9):1271-8; Martinez et al., Toxicol Sci.,2002 October; 69(2):409-23).

Interleukin-13 (IL13), a cytokine secreted by activated T lymphocytes,has been demonstrated to be both necessary and sufficient for theexpression of allergic asthma and for use in experimental models ofasthma, which include airway hyper responsiveness, eosinophilrecruitment, and mucus overproduction (Wills-Karp et al., Science, 1998;282:2258-2261). It has been shown, that selective neutralization of IL13will ameliorate the asthma phenotype (Grunig et al., Science, 1998;282:2261-2263). It has also been reported that IL13 is involved in theup regulation of mucin gene MUC8 expression in human nasal polypepithelium and cultured nasal epithelium (Kimm et al., ActaOtolaryngol., 2002; September;122(6):638-643; Seong et al., ActaOtolaryngol., 2002; June;122(4):401-407). MUC8, a major airway mucinglycoprotein, is implicated as playing a role in the pathogenesis ofmucus hypersecretion in chronic sinusitis with polps (Seong et al., ActaOtolaryngol., 2002; June;122(4): 401-407).

Functionally, IL13 signals through a receptor complex consisting of theinterleukin-13 receptor alpha-1 chain (IL13RA1) and IL-4 receptor alpha(IL4RA) (Daines and Hershey, J Biol. Chem., 2002; 22(12):10387-10393).It has also been shown, that the interleukin-13 receptor alpha-2(IL13RA2) binds IL13 with high affinity, but by itself (Daines andHershey, J Biol. Chem., 2002; 22(12):10387-10393). This receptor lacks,however, the cytoplasmic domain necessary for signaling and, therefore,is considered to be a decoy receptor. It has been shown that IL13RA2 ispredominately an intracellular molecule that can be quickly mobilizedfrom intracellular stores and surface expressed following cellulartreatment with interferon (IFN)-gamma. The surface expression of IL13RA2after IFN-gamma treatment does not involve protein synthesis and resultsin diminished IL13 signaling (Daines and Hershey, J Biol. Chem., 2002;22(12):10387-10393).

The results of the gene expression array analysis for IL-31 indicate theaction of IL-31 to be novel to that of IFN-gamma in that the IL-31treatment of lung epithelial derived cell lines resulted in asignificant increase of IL13RA2 gene expression. Thus, IL-31 treatmentcan be beneficial in cases where long-term up regulation of IL13RA2expression and down regulation of IL13 is desired such as in asthma,airway hyperactivity (AHR), and mucin regulation, including chronicsinusitis with polyps.

The bioactive antagonists or antibody conjugates described herein can bedelivered intravenously, intraarterially or intraductally,subcutaneously, topically, or may be introduced locally at the intendedsite of action.

Within an aspect, the invention provides a method of treating asthma,airway hyper-responsiveness, allergic rhinitis, and chronic obstructivepulmonary disease (COPD) comprising administering an IL-31 agonist to amammal. Within an embodiment the IL-31 agonist is selected from thegroup consisting of: a) a polypeptide of at least 70% sequence identityto the polypeptide of SEQ ID NO: 2 from residue 27 to residue 164; b) apolypeptide comprising the sequence of SEQ ID NO: 2 from residue 27 toresidue 164; c) analogues of b); d) derivatives of b); e) variants ofb): and f) fragments of b). Within an embodiment the inflammation isinhibited, minimized, prevented or neutralized.

The invention provides a method of treating asthma, airwayhyper-responsiveness, allergic rhinitis, comprising administering anIL-31 agonist to a mammal. In an embodiment, the IL-31 agonist isselected from the group consisting of: a) a polypeptide of at least 90%sequence identity to the polypeptide of SEQ ID NO: 2 from residue 27 toresidue 164; and b) a polypeptide comprising the sequence of SEQ ID NO:2 from residue 27 to residue 164. In an embodiment, inflammation isinhibited, minimized, or neutralized. Within an embodiment the IL-31agonist is produced in mammalian cells. In another embodiment the IL-31agonist is produced in E. coli. In an embodiment, the cysteine residuesof the amino acid sequence of the IL-31 agonist are mutated to producehomogenous preparations of IL-31.

Within an aspect the invention provides a method of treating asthma,airway hyper-responsiveness, allergic rhinitis, comprising administeringan IL-31 agonist to a mammal wherein the IL-31 agonist is administeredduring sensitization or challenge. Within an embodiment the IL-31agonist is not administered as a pre-treatment to the asthma, airwayhyper-responsiveness or allergic rhinitis. Within an embodimentproduction of proinflammatory cytokines in the lung and BAL fluid isinhibited, minimized, or neutralized. In an embodiment theproinflammatory cytokines are IL-5 or IL-13. In an embodiment theproinflammatory cytokines are IL-5 and IL-13.

Within an aspect the invention provides a method of inhibiting,minimizing, or neutralizing the production of proinflammatory cytokinesin the lung and BAL fluid in a pulmonary inflammatory condition,comprising administering a polypeptide wherein the polypeptide isselected from the group consisting of: a) a polypeptide of comprising atleast 90% sequence identity to the polypeptide of SEQ ID NO: 2 fromresidue 27 to residue 164; and b) a polypeptide comprising the sequenceof SEQ ID NO: 2 from residue 27 to residue 164. In an embodiment, thepolypeptide is produced in mammalian cells. In another aspect thepolypeptide is produced in E. coli.

The invention provides a method for optimizing the dose of an IL-31agonist used to treat asthma, airway hyper-responsiveness, or allergicrhinitis comprising determining the amount of the IL-31 agonist thatproduces a decrease in proinflammatory cytokines. In an embodiment, theproinflammatory cytokines are IL-5 or IL-13. In an aspect theproinflammatory cytokines are IL-5 and IL-13.

The invention provides a use of a pharmaceutical composition comprisingan IL-31 agonist to treat, minimize, reduce or inhibit the symptoms ofasthma, airway hyper-responsiveness or allergic rhinitis wherein thepharmaceutical composition is selected from the group consisting of: a)a polypeptide of comprising at least 90% sequence identity to thepolypeptide of SEQ ID NO: 2 from residue 27 to residue 164; b) apolypeptide comprising amino acid residues 27 to 164 of SEQ ID NO: 2.

The invention provides a kit for determining the optimum dose fortreating asthma, airway hyper-responsiveness or allergic rhinitiscomprising: a) taking a sample of lung tissue or BAL fluid from apatient with asthma, airway hyper-responsiveness or allergic rhinitis;b) testing the sample in vitro to determine if an amount of an IL-31agonist decreases proinflammatory cytokine production in the sample,wherein the amount of the proinflammatory cytokine is measured bydetermining the level of gene expression or protein; c) determining thedosage of the IL-31 agonist sufficient to reduce levels of theproinflammatory cytokine.

The invention provides a method of down-regulating the expression ofIL-31Ra in a condition such as asthma, airway hyper-responsiveness orallergic rhinitis comprising administering an amount of an IL-31agonist. In an embodiment, the IL-31 agonist is selected from the groupconsisting of: a) a polypeptide of at least 90% sequence identity to thepolypeptide of SEQ ID NO: 2 from residue 27 to residue 164; and b) apolypeptide comprising the sequence of SEQ ID NO: 2 from residue 27 toresidue 164. In an embodiment, inflammation is inhibited, minimized, orneutralized. In an embodiment., the IL-31 agonist is produced inmammalian cells. In another embodiment, the IL-31 agonist is produced inE. coli. In an embodiment the cysteine residues of the amino acidsequence of the IL-31 agonist are mutated to produce homogenouspreparations of IL-31.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Analysis of IL-31 in Airway Hyper-ResponsivenessMurine Model

A) Sensitization and Airway Challenge

Female BALB/c and C57B1/6 mice 6 weeks of age were purchased fromCharles River Laboratories and maintained under SPF conditions. Groupsof mice 8 to 10 wks of age, were sensitized by intraperitoneal injectionof 10 ug of OVA (Calbiochem) in 50% Imject Alum (Pierce) on days 0 and7. Seven days later, mice were challenged on 2 consecutive days (days 14and 15) with 20 ug of OVA in 50 ul PBS. Forty-eight hours followingallergen challenge whole lung tissue, BAL cellular infiltrates, BALfluid and serum from half the sensitized animals were collected forfurther analysis while the remaining mice were assessed for airway hyperresponsiveness (AHR).

B) Bronchoalveolar Lavage

Bronchoalveolar lavage fluid was collected via intratrachealcannulation. Saline was slowly injected in the lung and withdrawn in 4×1ml aliquots. The lavage fluids were centrifuged to isolate the BAL cellsand the supernatant was frozen for later analysis. BAL cell pellets wereresuspended at 2 million cells per ml and 150 ul was used for total anddifferential cell counts. Total BAL leukocyte counts were determined foreach mouse via light microscopy using trypan blue exclusion.Differential cell counts in the lavage fluid of each animal weredetermined by H&E staining (DiffQuik; Merz & Dade, Dubingen,Switzerland) of air-dried and fixed cytospin slides. Cell counts werecalculated by examining one hundred cells per cytospin (PhoenixLaboratories). The total number of different leukocytes was calculatedfrom the data collection. Results are expressed as number of cells perlung.

C) Measurement of Airway Hyper-Responsiveness

Airway responsiveness was assessed as a change in airway functionfollowing challenge with aerosolized methacholine (MCh) using whole-bodyplethysmography (Buxco, Electronics, Shannon, Conn.) 14. Briefly,unrestrained, conscious mice were placed in a whole-bodyplethysmographic chamber and respiratory waveforms were measured for 5min to obtain a basal line. After basal values were established, micewere challenged with aerosolized saline for the unchallenged controlmeasurement and then increasing concentrations of MCh (0.075M to 0.3 M).Readings were taken over a 10 min period 3 min after each nebulizationperiod. Data are expressed as fold increase above basal values using thedimensionless parameter PehnH.

D) RNA Isolation and Real-time TaqMan PCR Analysis

Lung tissue and BAL cells were collected from animals 48 h followingantigen challenge. Snap frozen whole tissue samples and BAL cellpellets, resuspended in RLT buffer, were stored at −80° C. untilprocessed for RNA isolation. Briefly, lung tissue was homogenized in RLTbuffer (Qiagen) and extracted using the commercially available RNeasykits as per the manufacturer's instructions (Qiagen, Valencia, Calif.).The RNA was transcribed into first strand cDNA using Taqman RT-PCRreagents (Applied Biosystems, Branchburk, N.J.), according to themanufacturer's protocol. Levels of murine IL-31, IL-31RA, IL-4, IFNg,TNFa, CD40, CD40L, Class II, Cathepsin L, IL-13Ra2, MIP-2, IL-8R,Eotaxin and OSMR mRNA were determined via multiplex real-time TaqManPCR. Oligonucleotide primers and TaqMan probes were designed using thePrimer Express software (PE Applied Biosystems, Foster City, Calif.) andwere synthesized in house. Forward primer, reverse primer and probesequences were generated. Levels of mRNA for each gene were calculatedrelative to the internal house-keeping gene,hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) using theComparative Ct method (User Bulletin #2, PE Applied Biosystems).

E) BAL Fluid and Serum Cytokine Analysis

Cytokine levels in BAL fluid supernatants and serum samples weremeasured using the Mouse Cytokine LINCOplex kit (LINCO Research, StCharles, Miss.) and the Luminex100 plate reader (Luminex Corporation,Austin, Tex.) according to the manufacturer's instructions.Quantification of cytokines was performed by regression analysis from astandard curve generated from cytokine standards included in the kit.Lower limits of detection for IL-5 and IL-13 were 0.6 pg/ml and 4.7pg/ml respectively.

F) IL-31 Administration by Osmotic Pump

Mouse IL-31 was delivered at a dose of 20 ug per day (approximately 1mg/kg per day) for 14 days by an osmotic minipump (Alzet) implantedsubcutaneously into the dorsum of BALB/c mice. PBS+0.1% BSA was includedas the vehicle control. Pumps were implanted on day 3 to ensure IL-31delivery throughout the course of the model.

G) Histopathology of Murine Lung

Lungs were fixed by inflation and immersion in 10% normal bufferedformalin (NBF).

Immunohistochemistry of Human Lung

5 uM sections were incubated with primary antibodies diluted from 333ng/ml to 1330 ng/ml for both IL31 and IL31RA for 60 min in ChemMateAntibody Dilution Buffer (part# ADB250, Ventana Medical systems).Tissues were washed twice in TBST, and then incubated for 45 min inbiotinylated goat anti-rabbit Ab, 750 ng/ml in PBSB (catalog #BA-1000,Vector Labs). Slides were washed and incubated in Vectastain Elite ABCReagent (catalog# PK-7100, Vector Labs) for 45 min and washed twice inTBST. Signals were developed with DAB+(catalog# K-3468, DakoCytomation)for 10 min at room temperature. Tissue slides were then counterstainedin hematoxylin (catalog# H-3401 Vector Labs), dehydrated andcoverslipped in VectorMount (catalog# H-5000, Vector Labs).

Statistical Analysis

Analysis of variance (ANOVA) was used to determine the levels ofdifference between groups for BAL differentials, and serum IgE.Student's t test was performed to determine differences between groupsfor gene expression studies. The data are expressed as mean±SD.Differences were considered statistically significant when p<0.05.

Results:

Systemic delivery with IL-31 during a mouse model of allergic asthmaresults in decreased levels of IL-5 and IL-13 mRNA and protein.Preliminary analysis of lung tissue and BAL cellular infiltrates fromanimals in a mouse model of antigen-induced asthma showed that mRNAencoding IL-31RA, the receptor for IL-31, was up-regulated in both wholelung tissue and lung cellular infiltrates 48 h after airway antigenchallenge (Dillon et al., 2004).

Purified IL-31 was delivered at 20 ug/day for 14 days during the courseof the allergen-induced asthma model via subcutaneous insertion of amini-osmotic pump. Forty-eight hours following allergen inhalation ofsensitized mice we collected whole lung tissue, BAL cellularinfiltrates, BAL fluid and serum. RNA isolated from lung tissue and BALcells were analyzed via quantitative TaqMan PCR for expression of 16genes, including IL-31RA, IL-31, IL-4, IL-5, IL-13, IFNg, TNFa, CD40,CD40L, Class II, Cathepsin L, IL-13Ra2, MIP-2, IL-8R, Eotaxin, and OSMR.Results from these studies showed significant down-regulation of IL-5 (p0.013), IL-13 (p 0.003) and Cathepsin L (p 0.038) mRNA in whole lungtissue, and decreases in IL-4 (p 0.01), IL-5 (p 0.003), IL-13 (p<0.001),Cathepsin L (p 0.007), Class II (p 0.005), CD40 (p 0.011), and CD40L(p<0.001) in BAL cell mRNA from allergen sensitized and challengedanimals treated with IL-31 compared to vehicle control treated animals.Analysis of BAL fluid for cytokines confirmed the down-regulation ofboth IL-5 and IL-13 (p<0.001). In addition, IL-31 treatment resulted inlower levels of IL-5 in the serum. Serum IL-13 could not be detected ineither the control or IL-31-treated animals.

IL-31 treatment results in decreased lung inflammation and airwayhyper-responsiveness following allergen sensitization and challenge. Theclassic triad of allergic asthma involves IgE production, airwayhyper-responsiveness (AHR) and eosinophilic inflammation. AHR is awell-established characteristic of allergic asthma and is believed to bethe result of airway mucosal inflammation. Clinical investigations havesuggested a relationship between the presence of activated airwayinflammatory cells, including T cells, mast cells, monocytes,eosinophils and neutrophils, morphologic changes in airway tissues, andthe development of severity of AHR (See Bradley B., et al., J. AllergyClin. Immuno1.88:661-74, 1991; and Wardlaw A. et al., Am. Rev. Respir.Dis. 137:62-9, 1998). Analysis of airway infiltrating cells followingallergen sensitization and challenge in the presence of daily IL-31treatment resulted in significant decreases in lymphocytes (p 0.001),macrophages (p0.029) and eosinophils (p 0.019) in BAL fluid.Histological analysis of formalin fixed lung tissue indicated thatinflammatory cell infiltrates and goblet cell hyperplasia in lungs frommice treated with IL-31 were substantially less than vehicle controls,suggesting a beneficial effect of IL-31 in airway inflammation.

Analysis of AHR of IL-31-treated animals via whole body plethysmographyindicated that mice treated with IL-31 exhibited less AHR compared tovehicle treated controls. No change was apparent in serum IgE levelsbetween IL-31- or vehicle-treated mice. These data therefore indicatethat IL-31 treatment decreases disease pathogenesis in a murine model ofallergic asthma, possibly through the down-regulation of IL-5 and IL-13.

IL-31RA is expressed in human alveolar macrophages, type II pneumocytesand bronchiolar epithelium. Immunohistochemical (IHC) analysis ofIL-31RA expression in human asthmatic lung and normal tissues indicatesIL-31RA is present on alveolar macrophages, type II pneumocytes (anepithelial derived cell type that is responsible for secretion ofsurfactant) and bronchiolar epithelium. Comparison between asthmaticlung and normal lung tissue showed no difference in the cellularstaining pattern.

Example 2 Human Monocyte Staining

Whole blood (200 ml) was collected from a healthy human donor and mixed1:1 with PBS in 50 ml conical tubes. Thirty ml of diluted blood was thenunderlayed with 15 ml of Ficoll Paque Plus (Amersham Pharmacia Biotech,Uppsala, Sweden). These gradients were centrifuged 30 min at 500 g andallowed to stop without braking. The RBC-depleted cells at the interface(PBMC) were collected and washed 3 times with PBS. The isolated humanPBMC yield was 300×10⁶ prior to selection described below.

The PBMCs were suspended in 3 ml MACS buffer (PBS, 0.5% BSA, 2 mM EDTA)and 1×10⁶ cells were set aside for flow cytometric analysis. We nextadded 0.45 ml anti-human CD14 microbeads (Miltenyi Biotec) and themixture was incubated for 20 min at 4 degrees C. These cells labeledwith CD14 beads were washed with 30 ml MACS buffer, and then resuspendedin 1.5 ml MACS buffer.

An LS column (Miltenyi) was prepared according to the manufacturer'sinstructions. The LS column was then placed in a MidiMACS magnetic field(Miltenyi). The column was equilibrated with 3 ml MACS buffer. The cellslabeled with anti-human CD14 microbeads were then applied to the column.The CD14-negative cells were allowed to pass through. The column wasrinsed with 10 ml (2×5 ml) MACS buffer and the rinse was pooled with theCD14-negative flow-through cells. The column was then removed from themagnet and placed in a 15 ml falcon tube. CD14-positive cells wereeluted by adding 5 ml MACS buffer twice to the column and bound cellsflushed out using the plunger provided by the manufacturer. The yield ofCD14+ selected human peripheral blood monocytes was 30×10⁶ total cells.One million of these monocytes were set aside for flow cytometricanalysis. The CD14-negative flow-through cells were counted and 1×10⁶cells were set aside for flow cytometric analysis.

The 1×10⁶ PBMCs, CD14-positive and CD14-negative cells that had been setaside were stained and run on a fluorescence activated cell sorter(FACS) to assess the purity of the CD14+ selected human peripheral bloodcells. A FITC-conjugated anti-human CD19 antibody, an anti-human CD56-PEAb, an anti-human CD11b-CyChrome Ab, and an anti-human CD3-APC Ab (allfrom PharMingen) were used for staining the cells. The CD14+ selectedcells were 88% CD14+. The PBMCs were 10% CD14+ and the CD14-negativecells were 0.1% CD14+.

The human CD14+ selected human peripheral blood monocytes were activatedby incubating them at 2×10⁶ cells/ml in RPMI+10% human ultraserum(Gemini Bioproducts, Calabasas, Calif.) with and withoutrhInterferon-gamma (IFNg) 10 ng/ml (R&D) for 4, 8, 12 or 24 hours at 37°C. in ultra low-attachment tissue culture plates (Corning/Costar). Ateach timepoint, the cells were harvested, pelleted, washed once withFACS stain buffer (PBS, 3% human ultraserum, 1% BSA, 10 mM HEPES) andcounted.

The activated monocytes were stained by FACS as follows: 1×10⁶ cellswere combined with specific and non-specific antibody blockingreagents-soluble receptor IL-31RaCEE and zVen1CEE respectively—at 200ug/ml or none. The cells were then combined with either 2.0 μg/mL ofbiotinylated mouse anti-human IL-31Ra or biotinylated mouse isotypenegative control (Southern Biotechnology) or left unstained for 30minutes on ice in FACS buffer. Cells were washed twice with FACS bufferand then stained with SA-PE (Jackson Immuno Laboratories) at 1:400 incombination with FITC-conjugated anti-human CD14 antibody at 1:100(PharMingen) for 20 minutes on ice. Cells were then washed twice withFACS buffer and resuspended in 400 ul FACS buffer containing7-aminoactinomycin D (Molecular Probes) at 1:800 and analyzed by FACS ona BD FACSCaliber using CellQuest software (Becton Dickinson, MountainView, Calif.).

The biotinylation of mouse-anti-human IL-31 Ra was done as follows: 205μL of mouse anti-human IL-31Ra (clone#276.100.5.5) at 2.45 mg/mL wascombined with 15 μL of 2 mg/mL EZ-link Sulfo-NHS-LC-biotin (Pierce,Rockford, Ill.) dissolved in ddH2O. This solution was incubated on arocker for 30 minutes at room temperature. After biotinylation thesolution was purified on a PD-10 column (Amersham Biosciences, Uppsala,Sweden).

Human CD14+ selected human peripheral blood monocytes activated withrhIFNg for 12 h and 24 h showed binding to the biotinylated mouseanti-human IL-31 Ra reagent+SA-PE. The binding was most pronounced atthe 12 h timepoint. This binding did not occur in cells initiallycombined with specific competitor protein IL-31. There was no stainingwith SA-PE alone or with the biotinylated mouse isotype negativecontrol+SA-PE. No binding was observed with the biotinylated mouseanti-human IL-31 Ra reagent+SA-PE on CD14+ selected human peripheralblood monocytes activated with rhIFNg for 4 h and 8 h.

Example 3 Effects of IL-31 on Allergen Induced AirwayHyper-Responsiveness

3.1 Materials and Methods

3.1.1 Mice.

Female BALB/c mice were purchased from Charles River Laboratories andmaintained under SPF conditions. All experimental animals used wereunder a protocol approved by the Institutional Animal Care and UseCommittee (IACUC) of ZymoGenetics. Mice were 11 weeks of age at onset ofstudy.

Tg mice over-expressing murine IL31, driven by the lymphocyte-specificpromoter/enhancer Eμ/1ck, or the ubiquitous promoter EF1α were used toevaluate the effects of IL-31 in vivo. The serum of EF1 μTg micecontained 0.3-1.1 ng/ml mIL-31, while the Eμ/1ck Tg serum contained10-43 ng/ml. Both types of IL-31 Tg mice develop a striking skinphenotype around 4-8 weeks of age, consisting of piloerection followedby mild to severe alopecia. The Tg skin is also highly pruritic, asevidenced by the scratching behavior of the mice, often excessive enoughto induce excoriation and lesions of the skin.

3.1.2 Sensitization and Airway Challenge.

Mice between the age of 8 and 12 weeks were sensitized by 100 □Lintraperitoneal injection of 10 ug of OVA (Calbiochem) in 50% ImjectAlum (Pierce) on days 0 and 7. One week later, mice were challengedintranasally on two consecutive days (days 14 and 15) with 20 □g of OVAin 50 uL PBS. Forty-eight hours following intranasal challenge with OVA,whole lung tissue, bronchoalveolar lavage (BAL) cellular infiltrates,BAL fluid and serum were collected from animals. In some experiments, anextra group of animals were put on protocol and tested for airwayhyper-responsiveness (AHR) by whole body plethysmography (WBP).

3.1.3 IL-31 Administration by Osmotic Pump.

Murine IL-31 was delivered for either 7 or 14 days by an osmoticmini-pump (Alzet) implanted subcutaneously into the dorsum of BALB/cmice. PBS+0.1% BSA was included as the vehicle control. The quantity ofIL-31 delivered is outlined below. In general, delivery of IL-31 with a14 day pump ensured that IL-31 was present in the circulation duringboth the allergen sensitization and challenges phases. The majority ofexperiments were performed with E. coli-derived IL-31 (SEQ ID NO: 17),however BHK-derived material (SEQ ID NO: 2) was shown to have a similareffect.

3.1.4 Measurement of Airway Hyper-Responsiveness.

Airway responsiveness was assessed as a change in airway functionfollowing challenge with aerosolized methacholine (MCh) using whole-bodyplethysmography (Buxco, Electronics, Shannon, Conn.). Briefly,unrestrained, conscious mice were placed in a whole-bodyplethysmographic chamber and respiratory waveforms were measured for 5min to obtain a basal line. After basal values were established, micewere challenged with aerosolized PBS for the unchallenged controlmeasurement and then increasing concentrations of MCh (0.075M to 0.3 M).Readings were taken over a 10 min period 3 min after each nebulizationperiod. Data are expressed as fold increase above basal values using thedimensionless parameter Penh (enhanced pause).

3.1.5 Bronchoalveolar Lavage.

Bronchoalveolar lavage fluid was collected via intratrachealcannulation. PBS with 0.5% FBS was slowly injected in the lung andwithdrawn in 3×1 ml aliquots. The lavage fluids were centrifuged toisolate the BAL cells and the supernatant was frozen for later analysis.BAL cell pellets were resuspended at 2 million cells per ml and 150□Lwas used for total and differential cell counts. Total BAL leukocytecounts were determined for each mouse via light microscopy using trypanblue exclusion. Differential cell counts in the lavage fluid of eachanimal were determined by H&E staining (DiffQuik; Merz & Dade, Dubingen,Switzerland) of air-dried and fixed cytospin slides. Cell counts werecalculated by examining one hundred cells per cytospin (PhoenixLaboratories). The total number of different leukocytes was calculatedfrom the data collection. Results are expressed number of total cellsper lung.

3.1.6 RNA Isolation and Real-Time Taqman PCR Analysis.

Lung tissue and BAL cells were collected from animals 48 h followingantigen challenge. Lung tissues from animals were analyzed separately,whereas BAL cells from animals within a group were pooled, due to thesmall amount of material. Snap frozen whole tissue samples and BAL cellpellets, resuspended in RLT buffer, were stored at −80° C. untilprocessed for RNA isolation. Briefly, lung tissue was homogenized in RLTbuffer (Qiagen) and extracted using the commercially available RNeasykits as per the manufacturer's instructions (Qiagen, Valencia, Calif.).The RNA was transcribed into first strand cDNA using TaqMan RT-PCRreagents (Applied Biosystems, Branchburk, N.J.), according to themanufacturer's protocol. Oligonucleotide primers and TaqMan probes weredesigned using the Primer Express software (PE Applied Biosystems,Foster City, Calif.) and were synthesized in house. Forward primer,reverse primer and probe sequences were prepared. Real-time PCR were runin triplicate in 384-well plates on ABI Prism 7900HT (AppliedBiosystems). Real-time data were acquired and analyzed using SDS 2.0software (Applied Biosystems) with manual adjustment of baseline andthreshold parameters. Levels of mRNA for each gene were calculatedrelative to the internal housekeeping gene,hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) using theComparative Ct method (User Bulletin #2, PE Applied Biosystems).

3.1.7 BAL Fluid Cytokine Analysis.

Cytokine levels in BAL fluid supernatants and serum samples weremeasured using a custom Mouse Cytokine LINCOplex kit (LINCO Research, StCharles, Miss.) and the Luminex100 plate reader (Luminex Corporation,Austin, Tex.) according to the manufacturer's instructions.Quantification of cytokines was performed by regression analysis from astandard curve generated from cytokine standards included in the kit.

3.1.8 Quantification of Serum IgE.

Serum levels of total IgE and OVA-specific IgE were measured by ELISA.ELISA microtiter plates (Nunc Maxisorb) were coated overnight with 100ul/well of 2 ug/ml capture anti-IgE (Pharmingen cat#553413) in PBS at 4°C. Plates were then blocked with 200 ul/well SuperBlock (Piercecat#37515) for 15 minutes RT, then washed with ELISA C. Diluted IgEstandards (Pharmingen cat#557079) in ELISA B (PBS, 1% BSA) were platedserial 2 fold dilutions from 500 ng/ml. Serum samples diluted 1:50 inELISA B were plated 100 ul/well. If measuring Ova-specific IgEconcentrations, sera from mice that have been immunized and boosted withova/alum was used as a positive reference and serum from naïve mice as anegative reference. Reference sera were diluted 1:50, same as samplesera. Plates were incubated overnight at 4° C. and then washed withELISA C. Biotinylated detection anti-mouse IgE (Pharmingen cat#553419)at 2 ug/ml in ELISA B was then plated 100 ul/well, and incubated 60minutes at RT. Washed plates in ELISA C, and then plated 100 ul/well ofSA-HRP (Pharmingen cat#554066) diluted 1:1000 in ELISA B, and incubated30 minutes at RT. After incubation, plates were washed with ELISA C,then developed using OPD (10 ml NaCitrate/citris acid pH5, 1 OPD tablet(Pierce, Cat#34006), 10 ul H2O2). Stopped development of ELISA platewith 0.1M H2SO4, and read on spectrophotometer at 490 nm.

Results

3.2.1 Expression of IL-31RA in Murine Airways

3.2.1.1 Regulation of IL-31RA and OSMR mRNA

In initial studies of OVA sensitized and challenged mice, mRNA from lungtissues and cells from BAL were analyzed for expression of IL-31receptor to determine the potential for IL-31 activity inallergen-induced airway inflammation. BALB/c and C57B1/6 mice weresensitized with OVA, then challenged intranasally with either OVA as theallergen or PBS as the control. Quantitative RT-PCR analysis of tissuessuggested that IL-31Ra was significantly up-regulated in both lungtissue (FIG. 1) and in BAL cell infiltrates following allergen-challengein sensitized mice. OSMR, the other subunit of the IL-31 receptor, wasalso found to be expressed in the lung, though expression did not appearto be regulated as a result of allergen sensitization. In contrast, inBAL cells OSMR levels were very low. These data suggest that IL-31signaling may play a role in the development of airway inflammationfollowing allergen sensitization.

3.2.1.2 IL-31RA Protein Expression

To determine where IL-31RA protein was expressed in murine lungs, wecollected lungs from animals that had been sensitized with OVA andchallenged intranasally with either OVA or PBS. We then analyzedexpression of IL-31RA by immunohistochemistry and found that the majorcell type expressing IL-31 appeared to be macrophages. We also notedstaining in large monocytic cells that may represent resident monocytes.This was especially appreciated in the lung from PBS treated animals inwhich minimal inflammatory infiltrate was observed. It was also notedthat the occasional positive staining macrophage was observed in thealveoli of these latter, “uninvolved” lungs.

3.2.2 Delivery of IL-31 in OVA-Induced Airway Hyper-Responsiveness

3.2.2.1 Experiment #1

To follow the observation that IL-31Ra was regulated duringantigen-induced airway inflammation in mice, we studied the effect ofIL-31 delivery on the development of airway inflammation. To this end,OVA-specific airway inflammation was generated in the presence orabsence of circulating murine IL-31 in BALB/c animals. Briefly, BALB/cmice were sensitized intraperitoneally with 10 ug of OVA in Alum on day0 and day 7. On day 3, five of the sensitized animals were implantedsubcutaneously with an osmotic mini-pump that delivered murine IL-31(BHK-derived) for 14 days at 20 ug of IL-31 per day (approximately 1mg/kg per day). This rate of delivery resulted in approximately 20 ng/mlof IL-31 in the serum. Another group of five animals were implanted withpumps containing PBS+0.1% BSA as the vehicle control. Animals were thenchallenged intranasally on day 14 and 15 with OVA. A third group ofanimals were sensitized with OVA but challenged with PBS and wereincluded as a baseline control (no inflammation). Forty-eight hoursafter the last intranasal challenge, tissues were collected foranalysis. Lungs were lavaged for analysis of BAL cell infiltrates andmRNA was prepared from BAL cell infiltrates as well as whole lunghomogenates for analysis of gene regulation. Serum was collected foranalysis of cytokines and IgE levels.

3.2.2.1.1 Lung and BAL mRNA Analysis

Analysis of gene expression in lung with IL-31 delivery suggests thatIL-31 can significantly decrease expression of genes that have beenshown to be involved in the development of asthma and pulmonaryinflammation, including IL-5, IL13 and Cathepsin L (p value 0.0137,0.003 and 0.0381, respectively, BSA-treatment versus IL-31-treatment)(Table 1). There was also a trend towards decreases in the expression ofIL-4, IL-31Ra, TNFa, CD40 and CD40L, though the results did not reachstatistical significance. Interestingly, there was a significantincrease in IL-8R gene expression following IL-31-treatment compared tovehicle-control animals and a trend towards increases in MIP-2, thoughthis was not significant. MIP-2, and KC, are functional homologues ofIL-8 in mice. MIP-2 and KC increase the functional activity ofneutrophils including ingestion and killing of bacteria. Theimplications of these findings for IL-8Rand MIP-2 are unclear but it isknown that IL-8R is expressed on neutrophils and is required forneutrophil chemotaxis.

TABLE 1 mRNA Levels in Total Lung Homogenates from Vehicle- andIL-31-Treated Mice ^(a)Lung Vehicle v IL-31 Gene Vehicle-TreatedIL-31-Treated PBS p Value IL-4 0.081 + 0.041 0.038 + 0.006 0.001 + 0.000NS (p = 0.0535) IL-5 0.756 + 0.192 0.407 + 0.157 0.097 + 0.040 p =0.0137 IL-13 0.236 + 0.043 0.099 + 0.026 0.002 + 0.001 p = 0.003 IL-31Ra1.276 + 0.597 0.605 + 0.332 0.053 + 0.008 NS (p = 0.0591) TNFa 0.566 +0.160 0.073 + 0.041 0.057 + 0.054 NS MIP-2 0.623 + 0.439  1.36 + 0.6660.410 + 0.201 NS IL-8R 3.148 + 0.452 7.998 + 4.20  3.595 + 0.489 p =0.0334 BCL-6 4.645 + 0.487 4.962 + 0.632 2.409 + 0.279 NS CCL27 0.566 +0.139 0.760 + 0.139 0.659 + 0.361 NS CCR10 0.418 + 0.052 0.569 + 0.3880.444 + 0.027 NS TSLP 1.844 + 0.344 1.982 + 0.310 1.844 + 1.029 NSCathepsin L 198.8 + 72.61 113.8 + 24.44 30.63 + 8.145 p = 0.0381 ClassII 23.10 + 7.489 18.02 + 8.432 10.73 + 1.605 NS CCL17 8.219 + 2.0299.857 + 3.746 1.667 + 0.210 NS IL-13Ra 0.604 + 0.468 0.568 + 0.8330.004 + 0.002 NS Eotaxin-1 15.43 + 2.221 16.09 + 1.072 1.246 + 0.134 NSIL-10 0.182 + 0.049 0.179 + 0.045 0.016 + 0.009 NS CD40 8.469 + 2.7455.830 + 1.366 3.501 + 0.294 NS CD40L 0.747 + 0.241 0.493 + 0.123 0.479 +0.054 NS ^(a)Data are represented as mean + standard deviation of fiveanimals per group. Statistical analysis was performed using an unpairedt-test comparing vehicle-treated groups with IL-31 treated animals. ThePBS groups represent baseline values when animals have not challengedwith OVA.

Analysis of gene expression in cells within the BAL showed genes thatwere similarly regulated to the lung tissues. IL-4, IL-5, IL-13,IL-31Ra, Cathepsin L, Class II, CCL17, IL-10, CD40, CD40L were allsignificantly down-regulated in mRNA from BAL cells pooled from animalswithin each group. Of note, IL-8R and MIP-2 showed a trend towardsincreased expression in the BAL cells, but the results were notstatistically significant (Table 2).

TABLE 2 mRNA Levels in BAL Cell Homogenates from BSA- and IL-31-TreatedMice BAL Vehicle v IL-31 Gene Vehicle-Treated IL-31-Treated PBS p ValueIL-4 0.056 + 0.017 0.010 + 0.001 ND p = 0.0101 IL-5 0.090 + 0.0150.027 + 0.008 0.018 + 0.012 p = 0.0032 IL-13 0.080 + 0.006 0.018 + 0.003ND p < 0.0001 IL-31Ra  2.28 + 0.179  1.41 + 0.227 0.008 + 0.006 p =0.0066 TNFa 0.469 + 0.073 0.542 + 0.035 2.166 + 0.249 NS MIP-2 3.782 ±0.312 4.605 ± 0.488 3.335 + 0.221 NS IL-8R 1.459 + 0.304  1.709 + 0.12440.161 + 0.037 NS BCL-6 1.983 + 0.694  0.652 + 0.0462 0.459 + 0.014 p =0.0295 CCL27 0.103 + 0.048 0.058 + 0.015 0.025 + 0.006 NS CCR10 0.202 +0.039 0.270 + 0.048 0.198 + 0.051 NS TSLP 0.043 + 0.031 0.076 + 0.0310.040 + 0.047 NS Cathepsin L 731.3 + 69.52 426.5 + 80.06 42.60 + 4.661 p= 0.0076 Class II 18.43 + 4.192 5.070 + 0.598 2.759 + 0.151 p = 0.0054CCL17 10.89 + 1.584 8.236 + 0.306 0.347 + 0.048 p = 0.0462 IL-13Ra0.039 + 0.017 0.013 + 0.004 ND NS Eotaxin-1 0.073 + 0.017 0.055 + 0.0170.018 + 0.013 NS IL-10 0.326 + 0.046 0.130 + 0.019 0.009 + 0.008 p =0.0024 CD40 0.493 + 0.116 0.190 + 0.022 0.045 + 0.015 p = 0.0122 CD40L0.780 + 0.028 0.214 + 0.100 0.060 + 0.012 p = 0.0007 ^(a)Data arerepresented as mean + standard deviation of five animals per group.Statistical analysis was performed using an unpaired t-test comparingvehicle-treated groups with IL-31 treated animals. The PBS groupsrepresent baseline values when animals have not challenged with OVA.

3.2.2.1.2 Serum Cytokines and IgE Levels

Analysis of serum cytokines showed significant decreases in circulatinglevels of IL-5 protein between vehicle-treated (110+15.7 pg/ml) andIL-31-treated mice (37+8.4 pg/ml) (p<0.0001). Although IL-6, IL-9,IL-10, IL-12, GM-CSF, MIP-1 and RANTES were detected, no differenceswere observed between treatment groups. IL-4 and IL-13 protein could notbe detected in the serum of mice in this experiment.

No significant difference in circulating total IgE or OVA-specific IgEwas noted between the two groups of animals.

3.2.2.1.3 BAL Differentials

Analysis of cellular differentials in the BAL showed significantdecreases in the number of total lymphocytes in the BAL of IL-31-treatedmice compared to vehicle-treated animals (p 0.0095) and a trend towardsdecreases in BAL eosinophils, though the differences were notstatistically significant. The increased IL-8R mRNA expression in thelung and BAL tissues suggested there may be an increase in neutrophilsin the BAL. Although there was a trend towards increased neutrophilnumbers following IL-31-treatment, the results were not statisticallysignificant due to the large variation within the groups and the smallnumber of cells. It should also be noted that 48 hr is the optimaltime-point for measurement of macrophage and eosinophil influx, notneutrophil infiltration. Analysis of earlier time-points for moreprecise assessment of neutrophil infiltration may be warranted.

3.2.2.2 Experiment #2

A repeat of Experiment #1 analyzing antigen-specific airwayhyper-responsiveness, was performed in the presence or absence ofcirculating murine IL-31 in BALB/c animals.

3.2.2.2.1 Lung and BAL mRNA Levels

Only a subset of genes was analyzed in this experiment compared toExperiment #1. These genes included IL-4, IL-5, IL-13, IL-13Ra2, IL31RA,Cathepsin L and TNFa. A complete summary of levels of gene expressionrelative to the house-keeping control gene, HPRT are given in Table 3.In this experiment, significant decreases in gene expression for IL-13and TNFa were observed for mice treated with IL-31 compared to vehiclecontrol animals. Although there was a trend towards decreases in IL-5,IL-31RA and Cathepsin L mRNA, three genes which showed significantdecreases in the first experiment, the differences between treatmentgroups were not statistically significant in this experiment. Theresults for the BAL mRNA are shown in Table 4 and show that similargenes to the first experiment were significantly down-regulated in thisexperiment upon IL-31-treatment. These genes include significantdown-regulation of IL-5, IL-31RA and Cathepsin L. IL-13 levels were alsosignificantly down-regulated but the levels of IL-13 mRNA detected forthis analysis were at the lower limit of detection.

TABLE 3 mRNA Levels in Total Lung Homogenates from BSA- andIL-31-Treated Mice ^(a)Lung ^(b)Vehicle v IL-31 Gene Vehicle-TreatedIL-31-Treated PBS p Value IL-4 0.502 + 0.278 0.360 + 0.089 0.010 + 0.009NS IL-5 1.345 + 0.480 0.807 + 0.347 0.204 + 0.06  NS (p = 0.0772) IL-130.084 + 0.046 0.033 + 0.017 ND p = 0.0492 IL-31Ra 2.178 + 0.607 1.849 +0.691 0.211 + 0.066 NS TNFa 2.028 + 0.427 1.355 + 0.435 1.749 + 0.924 p= 0.0388 Cathepsin L 534.7 + 208.8 349.2 + 81.03 240.1 + 19.82 NSIL-13Ra2 1.043 + 0.342 0.940 + 0.424 0.024 + 0.001 NS ^(a)Data arerepresented as mean + standard deviation of five animals per group.^(b)Statistical analysis was performed using a unpaired t-test comparingvehicle-treated groups with IL-31 treated animals. The PBS groupsrepresent baseline values when animals have not challenged with OVA. NS= not significant, ND = not detected

TABLE 4 mRNA Levels in BAL Cell Homogenates from BSA- and IL-31-TreatedMice ^(a)BAL ^(b)Vehicle v IL-31 Gene Vehicle-Treated IL-31-Treated PBSp Value IL-4 0.064 + 0.029 0.056 + 0.003 ND NS IL-5 0.106 + 0.0060.048 + 0.001 ND p < 0.0001 IL-13 0.009 + 0.001 0.002 + 0.001 ND p =0.0007 IL-31Ra 2.198 + 0.189 1.536 + 0.180 0.006 + 0.002 p = 0.0118 TNFa1.138 + 0.281 2.047 + 0.836 2.546 + 0.278 NS Cathepsin L 424.9 + 93.04150.9 + 20.04 31.61 + 2.99  p = 0.0076 IL-13Ra2 0.033 + 0.010 0.027 +0.011 ND NS ^(a)Data are represented as mean + standard deviation offive animals per group. ^(b)Statistical analysis was performed using anunpaired t-test comparing vehicle-treated groups with IL-31 treatedanimals. The PBS groups represent baseline values when animals have notchallenged with OVA. NS = not significant, ND = not detected

3.2.2.2.2 BAL Fluid Cytokines

Results of the analysis of cytokines in the BAL fluid are summarized inTable 5. Consistent with results for regulation of mRNA in the lung andBAL cells, analysis of protein levels in the BAL fluid show significantdecreases in IL-5 (p<0.0001) and IL-13 (p<0.0001). In addition,significant increases in KC (p 0.0332) and MCP-1 (p 0.007) were observedin mice treated with IL-31. Although KC mRNA expression was not testedin this experiment, the previous experiment had shown evidence for KCup-regulation following IL-31 treatment. The detection of protein in theBAL fluid supports the finding that IL-31-treatment up-regulates KClevels in the lung. Levels of GM-CSF, IFN-g, IL-10, IL-12, IL-1b, IL-4,IL-6, IL-9 and RANTES were undetectable. MIP-1a and TNFa were detectedbut no difference were observed between IL-31-treated and untreatedanimals (Table 5).

TABLE 5 Cytokine Levels in BAL Fluid from BSA- and IL-31-Treated Mice^(a)BAL Fluid Cytokines Vehicle v IL-31 Cytokine Vehicle-TreatedIL-31-Treated p Value IL-13 18.61 + 1.93  5.23 + 1.30 p < 0.0001 IL-539.37 + 2.59 15.62 + 2.60 p < 0.0001 KC 22.81 + 1.63 40.59 + 7.32 p =0.0032 MCP-1  3.11 + 1.72 10.46 + 1.89 p = 0.007 MIP-1a  6.11 + 0.59 7.17 + 1.68 NS TNFa  1.11 + 1.20  1.70 + 1.15 NS ^(a)Data arerepresented as mean + standard deviation of five animals per group.Statistical analysis was performed using an unpaired t-test comparingvehicle-treated groups with IL-31 treated animals. The PBS groupsrepresent baseline values when animals have not challenged with OVA.

3.2.2.2.3 Serum Cytokines and IgE Levels

Analysis of serum cytokines suggested no statistically significantdecreases in circulating levels of any of the cytokines detectedincluding IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, GM-CSF, MIP-1a andTNFa. IL-4 and RANTES protein could not be detected in the serum of micein this experiment.

No statistically significant difference in circulating total IgE orOVA-specific IgE was noted between the two groups of animals.

3.2.2.2.4 BAL Differentials

Differential analysis of cells in the BAL suggested, similar to thefindings in the first experiment, that IL-31 treatment resulted in asignificant decrease in infiltrating eosinophils and a trend towardsdecreases in lymphocytes, though in this case, the differences forlymphocytes were not statistically significant. In this experiment, theobserved increase in neutrophils with IL-31 treatment compared tovehicle control animals did reach significance only when tested in aunpaired t-tailed t-test (p=0.0203), and is consistent with the findingof increased KC in the BAL fluid. However, statistical analysis using atwo-way ANOVA, to analyze all data and all parameters together,suggested that the differences in neutrophils between the two groups wasnot statistically significant.

3.2.2.3 Experiment #3

Similarly to the previous two experiments, analysis of antigen-specificairway hyper-responsiveness was performed in the presence or absence ofcirculating murine IL-31M BALB/c animals.

3.2.2.3.1 Lung and BAL mRNA Levels

A complete summary of levels of gene expression relative to thehouse-keeping control gene, HPRT are given in Table 5. The significantdecreases observed in IL-4, IL-5, IL-13 and IL-31Ra gene expression inmice treated with IL-31 compared to vehicle controls is consistent withthe previous two experiments (Table 6). As seen in Experiment #1, therewas also a trend towards increases in IL-8R gene expression in theIL-31-treated group compared to vehicle-treated mice however thedifferences were not statistically significant.

The results for the BAL mRNA are shown in Table 6 and reflect theresults found for the lung. IL-31-treatment induces significantdown-regulation of IL-4, IL-5, IL-13, and IL-31RA and increases in IL-8RmRNA expression in the BAL cells (Table 7).

TABLE 6 mRNA Levels in Total Lung Homogenates from Vehicle- andIL-31-Treated Mice ^(a)Lung Vehicle v IL-31 Gene Vehicle-TreatedIL-31-Treated PBS p Value IL-4 0.402 + 0.081 0.259 + 0.069 0.004 + 0.001p = 0.0266 IL-5 1.677 + 0.521 0.617 + 0.081 0.172 + 0.022 p = 0.0054IL-13 0.483 + 0.205 0.036 + 0.047 ND p = 0.0040 IL-31Ra 6.306 + 2.0552.132 + 1.341 0.274 + 0.044 p = 0.0102 CCL17 44.18 + 18.62 55.13 + 26.474.72 + 1.89 NS IL-8R 56.59 + 18.81 130.13 + 83.79  177.1 + 46.82 NS (p =0.0942) IL-13Ra2 1.145 + 0.304 0.779 + 0.343 0.013 + 0.006 NS ^(a)Dataare represented as mean + standard deviation of five animals per group.Statistical analysis was performed using an unpaired t-test comparingvehicle-treated groups with IL-31 treated animals.

TABLE 7 mRNA Levels in BAL Cell Homogenates from BSA- and IL-31-TreatedMice ^(a)BAL Vehicle v IL-31 Gene Vehicle-Treated IL-31-Treated PBS pValue IL-4 0.426 + 0.021 0.344 + 0.015 ND p = 0.0055 IL-5 0.573 + 0.1760.188 + 0.019 0.009 + 0.004 p = 0.0195 IL-13 0.243 + 0.084 0.002 + 0.001ND p = 0.0076 IL-31Ra 18.89 + 5.21  7.496 + 1.629 0.079 + 0.011 p =0.0224 CCL17 84.89 + 7.725 99.12 + 9.007 4.557 + 0.266 NS IL-8R 39.35 +9.557 105.32 + 10.78  17.31 + 3.713 p = 0.0014 IL-13Ra2 0.143 + 0.0640.193 + 0.090 0.005 + 0.008 NS ^(a)Data are represented as mean +standard deviation of five animals per group. Statistical analysis wasperformed using an unpaired t-test comparing vehicle-treated groups withIL-31 treated animals.

3.2.2.3.2 BAL Fluid Cytokines

Results of the analysis of cytokines in the BAL fluid are summarized inTable 8. Similarly to the previous data, these data show significantdecreases in IL-5 (p=0.0133) and IL-13 (p<0.0001) with IL-31 Treatment.However, in contrast to the previous experiments, analysis of KC andMCP-1 in the BAL showed decreased levels in IL31-treated mice comparedto vehicle control animals (p=0.0405 and p=0.0387, respectively) (Table8). IL-1b, IL-10, MIP-1a and RANTES levels were below the limit ofdetection of the assay.

TABLE 8 Cytokine Levels in BAL Fluid from BSA- and IL-31-Treated Mice^(a)BAL Fluid Cytokines Vehicle v IL-31 Cytokine Vehicle-TreatedIL-31-Treated p Value IL-2 1.816 + 0.695 1.938 + 0.621 NS IL-4  3.19 +0.627 3.891 + 3.339 NS IL-5 80.38 + 21.96 47.49 + 18.11 p = 0.0133 IL-62.104 + 1.478 2.658 + 1.531 NS IL-9 47.47 + 14.79 36.33 + 4.002 NS IL-1212.76 + 2.038 13.25 + 2.976 NS IL-13 46.42 + 5.140 25.10 + 3.843 p <0.0001 IFNg 5.238 + 1.750 6.349 + 2.310 NS TNFa 8.026 + 1.882 8.126 +2.874 NS GM-CSF 26.27 + 5.496 29.72 + 4.761 NS MCP-1 19.79 + 3.02516.89 + 1.481 p = 0.0387 KC 15.43 + 5.737 9.969 + 2.819 p = 0.0405^(a)Data are represented as mean + standard deviation of five animalsper group. Statistical analysis was performed using an unpaired t-testcomparing vehicle-treated groups with IL-31 treated animals.

3.2.2.3.3 Serum Analysis

No significant difference in circulating total IgE or OVA-specific IgEwas noted between the two groups of animals. Serum cytokines were notmeasured

3.2.2.3.4 BAL Differentials

Differential analysis of cells in the BAL suggested that IL-31 treatmentresulted in a significant decrease in infiltrating lymphocytes(p=0.0011), macrophages (p=0.0291) and eosinophils (p=0.0198), as hasbeen observed in previous experiment. There was no statisticaldifference in the number of neutrophils found in BAL of mice in the twotreatment groups.

3.2.2.4 Experiment #4

In order to determine the overall effect of IL-31 treatment on airwayinflammation, this study was designed to include the collection of lungtissue for immunohistochemistry and to analyze the airwayhyper-responsiveness of live mice to allergen challenge by WBP. The micewere sensitized and challenged with OVA and treated with IL-31 (E.coli-derived) or vehicle as previously described in the last threeexperiments. Forty-eight hours the after the last intranasal challenge,tissue was collected for analysis as before. In addition, a portion ofthe lung was collected and preserved in 10% buffered neutral formalin orin Zn TRIS fixative. The formalin fixed tissue was processed, embeddedin paraffin, sectioned and the resulting slides stained with hematoxylinand eosin for microscopic evaluation.

3.2.2.4.1 Lung and BAL mRNA Levels

A complete summary of levels of gene expression relative to thehouse-keeping control gene, HPRT are given in Table 9. As expected, asignificant decrease in IL-5 and IL-31Ra gene expression was observed inmice treated with IL-31 compared to vehicle-treated controls (Table 9).There was a trend towards decreases in IL-4, IL-13 and Cathepsin L andsignificant decreases in IFNg and CD40L, which has been observedpreviously. In addition, we once again observe a statisticallysignificant increase in IL-8R expression in total lung homogenates(Table 9).

The gene expression levels in BAL cell infiltrates are summarized inTable 10 and indicate, similar to previous studies, that IL-31-treatmentinduces significant down-regulation of most of genes tested includingIL-4, IL-5, IL-13 and Cathepsin L. There was a trend towards decreasingIL-31Ra expression, though not statistically significant. In thisparticular analysis we found a significant increase is MIP-2 and TNFa,both of which have shown trends towards an increase in BAL cell mRNAfrom previous experiments (i.e. Experiment #1 and/or Experiment #2).IL-8R was also significantly increased in BAL mRNA, suggesting adisassociation between regulation of genes in the BAL cells and totallung.

TABLE 9 Levels in Total Lung Homogenates from Vehicle- and IL-31-TreatedMice ^(a)Lung Vehicle v IL-31 Gene Vehicle-Treated IL-31-Treated PBS pValue IL-4 0.042 + 0.027  0.02 + 0.014 0.001 + 0.000 NS IL-5 0.298 +0.056 0.097 + 0.048 0.029 + 0.010 p = 0.0016 IL-13 0.068 + 0.070 0.023 +0.016 ND NS IL-31Ra 0.713 + 0.129 0.352 + 0.263 0.047 + 0.015 p = 0.0484TNFa 0.245 + 0.094 0.167 + 0.031 0.342 + 0.121 NS MIP-2 0.570 + 0.1940.482 + 0.174 0.398 + 0.238 NS IL-8R 1.369 + 0.672 10.85 + 6.129 6.162 +1.876 p = 0.0337 IFNg 0.135 + 0.019 0.069 + 0.034 0.033 + 0.008 p =0.0153 CCL27 0.359 + 0.162 0.637 + 0.028 0.662 + 0.073 p = 0.0146Cathepsin L 86.49 + 39.68 37.90 + 33.67 18.59 + 1.48  NS (p = 0.0670)Class II 23.73 + 9.840 17.25 + 6.162 7.195 + 0.347 NS IL-13Ra ND ND NDND Eotaxin 5.696 + 2.416 5.073 + 3.232 0.317 + 0.057 NS IL-10 0.145 +0.086 0.112 + 0.076 0.005 + 0.003 NS CD40 3.343 + 0.559 2.562 + 0.8321.835 + 0.138 NS CD40L 0.576 + 0.165 0.301 + 0.137 0.183 + 0.071 p =0.0426 ^(a)Data are represented as mean + standard deviation of fiveanimals per group. Statistical analysis was performed using an unpairedt-test comparing vehicle-treated groups with IL-31 treated animals. ThePBS group represents baseline with no OVA challenge.

TABLE 10 mRNA Levels in BAL Cell Homogenates from Vehicle- andIL-31-Treated Mice ^(a)BAL Vehicle v IL-31 Gene Vehicle-TreatedIL-31-Treated PBS p Value IL-4 0.0867 + 0.005  0.011 + 0.002 ND p <0.0001 IL-5 0.188 + 0.005 0.054 + 0.013 ND p < 0.0001 IL-13 0.0352 +0.001  0.0026 + 0.001  ND p < 0.0001 IL-31Ra 1.719 + 0.760 0.671 + 0.2020.011 + 0.007 NS TNFa 0.293 + 0.045 0.582 + 0.085  5.38 + 0.404 p =0.0065 MIP-2 0.334 + 0.045 1.108 + 0.254  9.20 + 0.134 p = 0.0065 IL-8R 1.99 + 0.139 0.616 + 0.023  1.33 + 0.095 p < 0.0001 IFNg 0.196 + 0.0110.0681 + 0.006  0.003 + 0.001 p < 0.0001 CCL27 0.094 + 0.006 0.057 +0.02  0.030 + 0.003 p = 0.0385 Cathepsin L 851.6 + 40.64 284.5 + 20.8 46.07 + 10.03 p < 0.0001 Class II 27.69 + 3.34   5.41 + 0.476 1.265 +0.043 p = 0.0003 Eotaxin 0.011 + 0.003 ND ND ND IL-10 0.466 + 0.0510.075 + 0.008 0.004 + 0.001 p = 0.002 CD40 0.771 + 0.033 0.079 + 0.0100.024 + 0.002 P < 0.0001 CD40L 0.711 + 0.012 0.234 + 0.013 0.016 + 0.004p < 0.0001 ^(a)Data are represented as mean + standard deviation of fiveanimals per group. Statistical analysis was performed using an unpairedt-test comparing vehicle-treated groups with IL-31 treated animals. ThePBS group represents baseline with no OVA challenge.

3.2.2.4.2 Serum

Serum was analyzed for total and antigen-specific IgE in IL-31-treatedand vehicle-treated OVA-sensitized mice. In this particular experimentwe found a significant decrease in the level of OVA-specific IgE inIL-31 treated mice compared to vehicle-treated animals (p=0.0070).However, the differences in total IgE levels between the two treatmentgroups did not reach statistical significance.

Serum cytokines and cytokine levels in the BAL fluid were not analyzedin this experiment.

3.2.2.4.3 Lung Histology

Histological examination of the lungs from IL-31 treated and vehicletreated animals showed a number of microscopic changes associated withsensitization and challenge with OVA, including: 1) multifocal todiffuse peribronchial/perivascular subacute inflammation that wascharacterized by accumulations of eosinophils and/or neutrophils(segmented nucleus with eosinophilic cytoplasm) and large monocytic typecells along with some lymphocytes; 2) diffuse epithelial/goblet cellhyperplasia; and 3) multifocal macrophage infiltrate in the alveoli withsome multinucleated giant cell formation.

In some areas of the lung, there was evidence of perivascular edema withor without variable amounts of inflammatory infiltrates as well asevidence of interstitial fibrosis. When compared with the lungs fromvehicle-treated animals that were sensitized and challenged with OVA,the inflammatory changes in the IL-31-treated animals were substantiallyless severe suggesting a beneficial effect of IL-31. Representativeimages of lung from vehicle-treated and IL-31-treated animals are shownin.

The lung sections from vehicle-treated animals showing severeinflammation also showed an increased number of F4/80+ macrophageinfiltrating the alveoli, peribronchial and perivascular subacuteinflammatory area. Lung sections from IL-31-treated animal showed lessinflammation and also showed significant lower number of F4/80+macrophage infiltrate. Enumeration of F4/80+ macrophages showed that theF4/80+ cell density was significantly lower in IL31-treated animalscompared to BSA-treated mice (p=0.0154, Unpaired-T test).

OVA-sensitized animals were challenged intranasally and measured forairway hyper-responsiveness by whole body plethysmography. Analysis ofthe dimensionless parameter enhanced pause (PenH) following challengewith increasing concentration of methacholine suggested thatIL-31-treated animals were less sensitive to allergen challenge comparedto vehicle-treated mice. This data support the histopathology findingsand suggest that IL-31 treatment decreases airway inflammation toallergen.

BAL cell differentials were not performed in this experiment

3.2.2.5 Summary

The data presented suggest that IL-31 delivery during antigensensitization and airway challenge in mice results in down-regulation ofpulmonary inflammation, as assessed by histology and airwayhyper-responsiveness via whole body plethysmography. Analysis of generegulation in total lung homogenates and in mRNA from infiltrating cellsin the lung suggest that IL-31 can consistently down-regulate genes thathave been associated with pulmonary inflammation and asthma including,IL-5, IL-13 and Cathepsin L. Genes generally associated withinflammation were also found to be down-regulated and include IFNg, CD40and CD40L. The IL-31 effect on gene down-regulation appeared to be moreconsistent in mRNA from BAL infiltrating cells compared to total lungmRNA. Moreover, when cytokine levels in the BAL fluid were tested,concordant down-regulation of protein was often observed. Thisdown-regulation of Th2 and inflammatory genes often translated todecreases in BAL cell infiltrates, particularly eosinophils, macrophagesand lymphocytes. Analysis of F4/80+ macrophage cell numbers in lungs ofIL-31-treated animals via histomorphometry yielded quantitative datathat confirmed the decrease in tissue macrophages in the lung.Furthermore, analysis of general pathology associated with pulmonaryallergic inflammation showed decreases in severity of disease withdelivery of IL-31. These data therefore suggest that delivery of IL-31during allergen sensitization and challenge can reduce the severity ofpulmonary inflammation through an as yet unknown mechanism.

3.2.3 Dose Response to IL-31

2.3.1 Experiment #1

We next decided to investigate the minimum dose of IL-31 required forthe inhibitory effects on pulmonary inflammation following OVAsensitization and intranasal challenge in mice. In the first experiment,Alzet 14-day osmotic pumps were loaded with 10-fold decreasingconcentrations of IL-31, from 20 ug/day to 0.02 ug/day, and implanted inmice during OVA allergen sensitization and challenge as previouslydescribed. Five animals per group were analyzed.

3.2.3.1.1 Lung and BAL Cell mRNA

Previous studies have shown that delivering 20 ug/day of IL-31 decreasesexpression of a number of genes in the lung following allergensensitization and challenge. Results summarized in Table 11 show thatfor some of these genes, this effect of IL-31 is dose-dependant.Comparison of gene expression levels in the group of mice treated with20 ug/day of IL-31 with those receiving 10-, 100- or 1000-fold lessIL-31 shows significant differences. Genes including, IL-5, IL-13,IL-31Ra, TNFa, IFNg, Class II, IL-13Ra2, Eotaxin, IL-10, CD40 and CD40L,were more highly expressed at lower doses of IL-31, especially at the0.2 and 0.02 ug/day doses. Cathepsin L and IL-4 showed trends towardsincreased expression with lower doses of IL-31, but these trends werenot significant. Furthermore, IL-8R and MIP-2, which have been shown tobe increased following 20 ug/day IL-31-treatment, also showedsignificant IL-31-dose dependency.

TABLE 11 mRNA Analysis of Lungs from IL-31-Treated Mice ^(a)IL-31Concentration in pump Gene ^(b)20 ug/day 2 ug/day 0.2 ug/day 0.02 ug/dayIL-4 0.025 + 0.017 0.047 + 0.025 0.041 + 0.006  0.042 + 0.005 IL-50.094 + 0.033 0.195 + 0.115  0.292 + 0.131**  0.251 + 0.035* IL-130.012 + 0.007  0.057 + 0.030* 0.036 + 0.016   0.077 + 0.041** IL-31Ra0.294 + 0.157 0.543 + 0.208 0.826 + 0.312*  0.988 + 0.287** TNFa 0.340 +0.082 0.370 + 0.088 0.432 + 0.0804  0.656 + 0.204** MIP-2 12.25 + 9.78 4.942 + 4.497 6.436 + 1.050  4.555 + 2.648 IL-8R 6.943 + 2.740 5.498 +2.103 3.236 + 1.412*  3.477 + 0.914* IFNg 0.084 + 0.026 0.150 + 0.0520.279 + 0.154* 0.229 + 0.125 CCL27 1.114 + 0.184  0.834 + 0.130* 0.993 +0.152  0.914 + 0.129 Cathepsin L 49.32 + 12.92 68.84 + 14.57 98.11 +49.52  78.10 + 31.41 Class II 30.70 + 7.802 41.56 + 7.701 50.62 + 8.797* 52.79 + 31.41** IL-13Ra2 0.068 + 0.044 0.095 + 0.035 0.116 + 0.026  0.172 + 0.032** Eotaxin 7.820 + 3.676  13.69 + 2.827** 9.327 + 1.638  12.10 + 1.613* IL-10 0.057 + 0.032  0.110 + 0.018* 0.105 + 0.011* 0.108 + 0.039* CD40 5.606 + 1.180 5.616 + 1.180  9.194 + 1.910**6.772 + 2.000 CD40L 0.734 + 0.335 0.790 + 0.182 1.899 + 1.343* 1.308 +0.169 ^(a)Data a presented as mean + standard deviation of five animalsper group. ^(b)**p < 0.01 and *p < 0.05 using one-way ANOVA withDunnett's post test, comparing IL-31-treatment at lower concentrations(2, 0.2 and 0.02 ug/day) to treatment the standard 20 ug/day used inprevious experiments.

TABLE 12 mRNA Levels in BAL Cell Homogenates from Dose curveIL-31-Treated Mice ^(a)IL-31 Concentration in pump (ug/day) Gene ^(b)20ug/day 2 ug/day 0.2 ug/day 0.02 ug/day IL-4 0.039 + 0.009 0.031 + 0.007 0.055 + 0.009  0.049 + 0.005  IL-5 0.016 + 0.002 0.034 + 0.003*  0.080 +0.010** 0.082 + 0.006** IL-13 0.001 + 0.000 0.005 + 0.000**0.023_0.001** 0.015 + 0.000** IL-31Ra 0.850 + 0.250 1.286 + 0.079* 1.605 + 0.129** 1.420 + 0.100** TNFa 0.446 + 0.013 0.351 + 0.034* 0.282 + 0.039** 0.342 + 0.030** MIP-2 5.041 + 1.058 4.983 + 0.582 1.771 + 0.212** 1.304 + 0.095** IL-8R 4.269 + 0.308 2.244 + 0.246**2.678 + 0.138** 3.114 + 0.202** IFNg 0.072 + 0.012 0.162 + 0.020**0.242 + 0.019** 0.288 + 0.040** Cathepsin L 109.0 + 15.09 75.35 +5.911** 172.8 + 30.58  103.1 + 6.720  Class II 2.148 + 0.142 4.676 +0.407** 9.186 + 0.519** 10.24 + 1.293** IL-13Ra2 ND ND ND ND IL-100.055 + 0.004 0.084 + 0.007*  0.124 + 0.031** 0.110 + 0.002** CD400.066 + 0.008 0.071 + 0.006  0.124 + 0.006** 0.171 + 0.027** CD40L0.151 + 0.027 0.107 + 0.006  0.267 + 0.090  0.215 + 0.054  ^(a)Data apresented as mean + standard deviation of five animals per group.^(b)**p < 0.01 and *p < 0.05 using one-way ANOVA with Dunnett's posttest, comparing IL-31-treatment at lower concentrations (2, 0.2 and 0.02ug/day) to treatment the standard 20 ug/day used in previousexperiments.

3.2.3.1.2 BAL Fluid Differentials

Analysis of BAL cell infiltrates also suggested that there is a dosedependant effect on the quantity and type of cellular infiltratesfollowing intranasal challenge with allergen. Doses of IL-31 at 20 and 2ug/day are more effective at inducing significant decreases ineosinophil, macrophage and lymphocyte numbers in the lung cellinfiltrates compared to the lower doses of IL-31 (0.2 and 0.02 ug/day).

No statistical significance was observed in OVA-specific or total IgE(data not shown)

Serum, BAL fluid cytokines and airway hyper-responsiveness was notmeasured in this experiment.

3.2.3.2 Experiment #2

In the previous experiment, we demonstrated that the effect of IL-31 onairway inflammation was dose-dependant. Analysis of gene expression andBAL cell infiltrates suggested that a 10-fold lower dose of IL-31 (2ug/day) was able to significantly inhibit the level of cellularinfiltrate in the lung following intranasal challenge with the allergen.This experiment was designed to determine whether 2 ug/day and lowerdoses of IL-31 could decrease airway hyper-responsiveness followingallergen sensitization and challenge.

Alzet 14-day osmotic pumps were loading with increasing concentrationsof IL-31, from 0.005 ug/day to 2 ug/day, and implanted in mice duringOVA allergen sensitization and challenge. Analysis was performed aspreviously described on 5 mice per group.

3.2.3.2.1 Lung and BAL Cell mRNA

Analysis of lung mRNA from IL-31-treated animals, to determine thelowest effective concentration of the IL-31 for down-regulation of genesin the lung, demonstrated that although some genes like IL-4,1′-13,IFNg, IL-31Ra and TNFa showed a trend towards down-regulation ofexpression at increasing concentrations of IL-31 (maximum dose of 2ug/ml), the differences were not statistically significant in most genes(Table 14). CD40L was the only gene tested that showed significantdown-regulation of expression when only 2 ug or 0.2 ug/day of IL-31 wasdelivered. The PCR data for all genes tested in lung tissues aresummarized in (Table 13).

TABLE 13 mRNA Analysis of Lungs from IL-31-Treated Mice

^(a)Data a presented as mean + standard deviation of five animals pergroup. Statistical analysis was a one-way ANOVA with Dunnett's posttest, comparing IL-31-treatment versus vehicle control (no IL-31, greycolumn) (**p < 0.01 and *p < 0.05).

Table 14 shows the gene analysis for cells in the BAL fluid. Trends forgene regulation in the cells of the HAL were less obvious (Table 14).Consistent with previous studies, MIP-2 and IL-8R were up-regulated at 2ug/ml but not at lower concentrations of IL-31 (Table 14).Interestingly, there appeared to be an up-regulation of some genes atsome of the concentrations of IL-31 below 2 ug/day, suggesting apossible bell curve for IL-31 activity. This was particularly obviousfor IL-5 and IL-13 (Table 14). The corresponding protein levels in theBAL fluid demonstrate a consistent pattern between gene regulation andprotein levels in the BAL. KC, which is related to MIP-2, was also foundto be up-regulated at 2 ug/ml of IL-31.

TABLE 14 mRNA Levels in BAL Cell Homogenates from Dose curveIL-31-Treated Mice

^(a)Data are presented of mean + standard deviation of triplicate wellsof pooled BAL cells collected from five animals per group. Statisticalanalysis was performed using one-way ANOVA with Dunnett's post test,comparing IL-31-treatment to vehicle control treated animals (no IL-31,grey column) (**p < 0.01 and *p < 0.05).

3.2.3.2.2 BAL Fluid Cytokines

BAL fluid was analyzed for cytokines by a luminex multiplex assay. IL-4,IL-5. IL-9, IL-13 and KC were all detected in the BAL fluid. The levelsof cytokines in the BAL fluid appeared to reflect the data collected forexpression of the genes in the BAL, with the lower concentrations ofIL-31 inducing significantly higher levels of IL-5 and IL-13 protein.IL-4 also appeared to be up-regulated at the lower concentrations butthe difference between IL-31 treatment and no treatment did not reachstatistical significance. KC was found to be up-regulated at 2 ug/daycompared to no treatment. No TNFa, IFNg and MCP-1 was detected in theBAL fluid. MIP-2 was not tested.

3.2.3.2.3 BAL Fluid Differentials

Analysis of BAL differentials shows that there was a trend towards adecrease in eosinophil infiltrates in the BAL of mice treated with themaximum concentration of IL-31 in this study (2 ug/day) compared tothose mice that were treated with the lower doses. Due to the largedegree of variation, the data was not statistically significant. Therewas a significant decrease in macrophage numbers with 2 ug/day of IL-31.None of the other IL-31 concentrations seemed to be effective atdecreasing macrophage numbers.

3.2.3.2.4 Serum IgE

Past experiments have indicated that the decreases in inflammatoryparameters associated with IL-31 treatment at higher concentrations (20ug/day) generally do not result in decreases in either total IgE orOVA-specific IgE (refer to section 1 of this report). Nevertheless,serum was analyzed for total and OVA-specific IgE following treatmentwith different concentrations of IL-31. In this experiment, althoughthere were no differences in the levels of total IgE in the sera ofthese mice, there did appear to be an increase in OVA-specific IgE inthe groups that were treated with 0.02 and 0.01 ug per day of IL-31compared to either no IL-31 treatment or the lowest dose of IL-31(0.0054 day). It is interesting to note here that 0.1 ug/day of IL-31induced significant up-regulation of IL-5 and IL-13 mRNA and protein inBAL fluid (Table 15). This data might suggest that although highconcentrations of IL-31 decrease pulmonary inflammation to airwayallergens, low concentrations of IL-31 may have the opposite effect.

3.2.3.2.5 Whole Body Plethysmography

Mice were further analyzed by whole body plethysmography for airwayhyper-responsiveness following intranasal challenge with OVA. IL-31treatment at the concentrations tested in this experiment did notdecrease airway hyper-responsiveness.

3.2.3.3 Experiment #3

Our data have so far indicated that the anti-inflammatory effect ofIL-31 in a murine model of airway hyper-responsiveness requires deliveryof at least 20 ug/day of IL-31. A 10-fold decrease in IL-31concentration does not consistently reduce airway inflammation and doesnot result in reduce airway hyper-responsiveness to allergen challenge.Immunohistochemistry of the mouse lung indicates that the majority ofIL-31RA is expressed on both resident and infiltrating macrophage in thelung and it is unknown at this point if IL-31 is acting directly onthese cells to produce the anti-inflammatory effect or if there isanother target cell type. In order to determine if IL-31 acts on aresident cell type to down-regulate that inflammation associated withantigen-challenge and sensitization we wished to determine whether IL-31pre-treatment (IL-31 delivery prior to sensitization and challenge)would have the same effect as IL-31 delivery throughout both thesensitization and challenge phases of the model.

Mouse IL-31 was delivered at a dose of 20 □g per day (approximately 1mg/kg per day) by an osmotic mini-pump (Alzet) implanted subcutaneouslyinto the dorsum of BALB/c mice. Mice were either implanted with 7-daypumps 7 days prior to the first OVA sensitization (IL-31 pre-treatment)or with 14-day pumps three days post-sensitization (IL-31 treatment) tocompare treatment prior to sensitization and treatment duringsensitization and challenge. As in all experiments so far, PBS+0.1% BSAwas included as the vehicle control. Murine IL-31 derived from E. coli,was used to prepare IL-31 pumps.

3.2.3.3.1 Lung and BAL mRNA

Analysis of gene expression in total lung homogenates fromvehicle-treated, IL-31 pre-treated and IL-31 treated mice are summarizedin Table 15. Statistical analysis was performed by comparing the IL-31groups (pre-treatment or treatment) to the vehicle control group.

The data clearly show that down-regulation of genes associated withinflammation mostly only occurs when IL-31 treatment is presentthroughout sensitization and challenge. Genes requiring the presence ofIL-31 during the entire period of sensitization and challenge includeIL-4, IL-5 and IL-31RA. A number of additional genes were down-regulatedregardless of whether IL-31 was given as a pre-treatment or duringsensitization and challenge. These genes include IL-9, TNFa, IFNg,Cathepsin L, IL-10, CD40 and CD40L.

Interestingly, IL-13 was found to be significantly increased compared tovehicle treated controls when IL-31 was given as a pre-treatment (Table15). Of all the genes tested, IL-13 was the only gene that showed thispattern of expression. IL-13 has been shown to be involved in themanifestation of disease in this model.

TABLE 15 Levels in Total Lung Homogenates from Vehicle- andIL-31-Treated Mice IL-31 Pre- Gene Treatment IL-31 Treatment VehicleControl IL-4 0.041 + 0.009 0.0270.009* 0.048 + 0.012 IL-5 0.357 + 0.0740.242 + 0.172** 0.557 + 0.156 IL-9  0.009 + 0.007** 0.003 + 0.002**0.112 + 0.075 IL-13  0.207 + 0.055* 0.082 + 0.038  0.100 + 0.052 IL-31Ra1.452 + 0.433 0.612 + 0.243**  2.034 + 0.6116 TNFa  0.219 + 0.049*0.229 + 0.083*  0.439 + 0.174 MIP-2 1.199 + 0.586 2.748 + 0.795  1.990 +0.679 IL-8R 2.334 + 0.828 4.306 + 1.951  2.533 + 0.700 IFNg  0.158 +0.055** 0.125 + 0.058** 0.586 + 0.263 CCL27  0.474 + 0.56** 0.687 +0.061  0.823 + 0.170 Cathepsin  14.82 + 3.574** 13.537 + 5.252** 29.53 + 10.56 L Class II 54.04 + 9.134 45.30 + 12.71  46.16 + 6.801IL-13Ra2 ND ND ND Eotaxin 5.060 + 1.166 4.205 + 2.007  3.648 + 0.654IL-10  0.106 + 0.018* 0.067 + 0.027** 0.196 + 0.079 CD40  2.121 +0.293** 1.906 + 0.319** 3.531 + 0.692 CD40L  0.773 + 0.190* 0.721 +0.208*  1.245 + 0.421 ^(a)Data a presented as mean + standard deviationof five animals per group. **p < 0.01 and *p < 0.05 using one-way ANOVAwith Dunnett's post test, comparing IL-31 pre-treatment orIL-31-treatment with vehicle control (no IL-31).

Previous studies of gene regulation in mRNA from BAL cells has shownthat IL-31 treatment can prevent the up-regulation of mRNA encodingasthma related genes such as IL-5 and IL-13. In this experiment, similarresults were observed for IL-31 treated animals compared to vehicletreated controls (Table 16).

Once again, IL-31 treatment, when given during sensitization andchallenge, significantly down-regulated IL-5, IL-13, IL-31Ra, IFNg,Cathepsin L, Class II, CD40 and CD40L gene expression in cells collectedfrom the BAL. In striking contrast however, IL-31 pre-treatment appearsto significantly increase the expression of many of these genes whencompared to vehicle control animals including IL-5, IL-13, IFNg, ClassII, CD40 and CD40L.

These data suggest that IL-31 pre-treatment may have an adverse effecton pulmonary inflammation compared to vehicle control mice and mayindeed exacerbate the disease. We have seen in the previous experimentsthat IL-31 treatment down-regulates the expression of IL-31RA in bothlung and BAL cellular infiltrates. IL-31 pre-treatment may down-regulatethe receptor prior to sensitization so that Th2 responses areexacerbated.

Of note, IL-31 treatment up-regulated IL-8R and MIP-2 as has beenpreviously observed, whereas IL-31 pre-treatment had no effect.

TABLE 16 Levels in BAL mRNA Homogenates from BSA- and IL-31-Treated MiceIL-31 Pre- Gene Treatment IL-31 Treatment Vehicle Control IL-4 0.019 +0.004 0.009 + 0.002  0.013 + 0.004 IL-5  0.127 + 0.021** 0.027 + 0.008* 0.069 + 0.019 IL-9 ND ND ND IL-13  0.139 + 0.018** 0.024 + 0.004* 0.051 + 0.009 IL-31Ra 2.569 + 0.328 0.971 + 0.139** 2.459 + 0.131 TNFA0.260 + 0.021 0.433 + 0.041  0.274 + 0.014 MIP-2 1.563 + 0.059 4.110 +0.231** 0.994 + 0.081 IL-8R 0.671 + 0.026 1.442 + 0.143** 0.605 + 0.100IFNg  0.336 + 0.022** 0.118 + 0.015** 0.245 + 0.016 CCL27 0.059 + 0.0090.045 + 0.013  0.062 + 0.021 Cathepsin 60.79 + 4.704 43.76 + 4.628**83.75 + 20.88 L Class II  54.09 + 5.045** 10.173 + 0.608**  37.65 +1.466 IL-13Ra2 ND ND ND Eotaxin ND ND ND IL-10 0.298 + 0.071 0.071 +0.010** 0.221 + 0.029 CD40  0.202 + 0.023** 0.053 + 0.001** 0.137 +0.013 CD40L  0.793 + 0.021** 0.253 + 0.009** 0.590 + 0.080 ^(a)Data arepresented of mean + standard deviation of triplicate wells of pooled BALcells collected from five animals per group. **p < 0.01 and *p < 0.05using one-way ANOVA with Dunnett's post test, comparing IL-31pre-treatment or IL-31-treatment to vehicle control treated animals (noIL-31)

3.2.3.3.2 BAL Fluid Cytokines

The animals receiving IL-31 treatment showed a trend towards decreasesin IL-4, IL-5 and IL-13 in the BAL fluid compared to the vehicle treatedgroup, though the results were not statistically significant. Consistentwith previous studies with IL-31-treated mice, this group also hadsignificantly elevated KC levels. All other cytokines that were assayedwere below the limit of detection.

In contrast to the IL-31 treated animals, cytokine analysis of BAL fluidfrom mice that were pre-treated with IL-31 showed significant increasesin the levels of IL-5, IL-9, and IL-13 compared to vehicle treated mice.This data is consistent with the up-regulation of genes like IL-5 andIL-13 in mRNA from BAL cells following pre-treatment with IL-31 andsuggest that IL-31 pre-treatment may exacerbate pulmonary inflammation.

3.2.3.3.3 BAL Fluid Differentials

Analysis of cell differentials showed a trend towards lower eosinophilinfiltrates in the BAL of mice treated with IL-31 during OVAsensitization and challenge, which is consistent with previous studies.Pre-treatment of mice with IL-31 prior to sensitization and challengedid not appear to alter the type or number of cellular infiltratessignificantly from vehicle-treated control animals.

3.2.3.3.4 Serum IgE

There was no significant difference between groups in either total IgE,or OVA-specific IgE levels.

3.2.3.3.5 Whole Body Plethysmography

Groups of mice that were pre-treated with IL-31, treated with IL-31during sensitization and challenge, or treated with vehicle wereanalyzed for their sensitivity to airway hyper-responsiveness followingintranasal allergen challenge. Results indicate that IL-31 pre-treatmentdoes not affect airway hyper-responsiveness compared to a no IL-31treatment control. In contrast however, IL-31 treatment throughoutsensitization and challenge decreased airway hyper-responsiveness tolevels comparable to those animals that had not received an allergenchallenge.

This current study indicates that IL-31 pre-treatment is unsuccessful atdecreasing airway hyper-responsiveness and although pre-treatment mayincrease IL-5, IL-13 and IL-9 levels in the BAL fluid, this does notappear to exacerbate airway hyper-responsiveness compared to no IL-31treatment.

3.2.3.4 Summary

We have previously established that 20 ug/day of IL-31 delivered duringOVA sensitization and challenge results in down-regulation of pulmonaryinflammation. In this section we demonstrate that this effect isdose-dependent. Although concentrations of IL-31 as low as 2 ug/day canstill reduce expression of genes that are important in the developmentof airway hyper-responsiveness, this low concentration was notsufficient to result in reduced airway hyper-responsiveness as measuredby whole body plethysmography.

In addition, although the receptor for IL-31 appears to be expressed onboth resident and infiltrating macrophages in the lung, pre-treatmentwith IL-31 immediately prior to allergen sensitization is not sufficientto achieve the anti-inflammatory effects of IL-31 we have so farobserved. Moreover, it is possible that IL-31 pre-treatment may increaseproduction of Th-2-type cytokines such as IL-5, IL-13 and IL-9. Thisfinding may be in support of a recent finding suggesting that IL-31RAregulates Th-2-type inflammatory responses and that in the absence ofIL-31RA receptor Th-2-type responses may be exacerbated (3). We knowthat delivery of IL-31 can down-regulate IL-31RA and we postulate thatpre-treatment with IL-31 results in the effective absence of IL-31RAprior to antigen sensitization resulting in increased expression ofIL-5, IL-13 and IL-9. In this particular experiment however, thoseincreases in Th-2-type cytokines did not result in exacerbated airwayhyper-responsiveness.

3.2.4 Airway Hyper-Responsiveness in IL-31 Transgenics

3.2.4.1 Experiment #1

IL-31 transgenic animals were sensitized and challenged with OVA aspreviously described and tested for airway hyper-responsiveness by wholebody plethysmography. The data suggest that IL-31 transgenic animalswere significantly less sensitive to OVA-induced airwayhyper-responsiveness compared to wildtype control littermates.

3.2.4.2 Experiment #2

This experiment was to repeat the test of the sensitivity of IL-31transgenic mice to allergen induced airway hyper-responsiveness.Analysis of the data show similar findings to the previous experiment.IL-31 transgenic animals appear less susceptible to pulmonaryinflammation induced by a sensitizing allergen, especially at thehighest dose of methacholine.

3.2.4.3 Experiment #3

Having established that IL-31 transgenic animals appear less sensitiveto airway hyper-responsiveness as measured by whole bodyplethysmography, we analyzed the nature of the decreased inflammation inthe lungs. Therefore, IL-31 transgenic animals and wildtype controlswere sensitized with OVA and challenged via the airways with either OVAor PBS. Forty-eight hours following intranasal challenge, BAL fluid wascollected for assessment of lung cellular infiltrates and lung wascollected for mRNA and gene expression analysis. Serum was alsocollected for analysis of total and OVA-specific IgE. In thisexperiment, cytokines in the BAL fluid and serum were not measured.

3.2.4.3.1 Lung and BAL mRNA

Lung mRNA was tested by quantitative RT-PCR for expression of a panel ofgenes that have been implicated in pulmonary inflammation. Two sets ofcomparisons were made between the four groups of animals. Table 17 showsdata from the first analysis where IL-31 transgenic animals weredirectly compared to wildtype mice under conditions of either OVA or PBSsensitization. In general, there were no differences between IL-31transgenic and control littermates when challenged with either OVA orPBS. For a number of genes there appeared to be lower expression in thelungs of IL-31 transgenics following OVA sensitization including IL-4,IL-5, IL-13 and IL-31RA, however these were not statisticallysignificant (Table 17). One gene, IL-10, showed significantly higherexpression in the lung of wildtype mice following OVA challenge,compared to IL-31 transgenic mice.

TABLE 17 mRNA Levels in Total Lung Homogenates from IL-31 Transgenicmice following Sensitization and Challenge with OVA - Comparison ofWildtype versus IL-31 Transgenic ^(a)OVA Challenge ^(a)PBS Gene IL-31 TgWildtype IL-31 Tg Wildtype IL-4 0.020 0.041 0.001 0.001 IL-5 0.188 0.3000.105 0.093 IL-13 0.365 0.645 0.001 0.001 IL-31Ra 0.601 1.023 0.1790.192 IFNg 0.318 0.324 0.110 0.136 TNFa 0.973 0.654 0.797 0.693 MIP-20.946 1.878 1.302 0.539 IL-8R 3.666 4.136 4.205 3.696 CCL27 0.511 0.5280.806 0.814 Cathepsin L 34.840 43.339 45.421 55.763 Class II 49.18050.207 24.050 33.128 IL-13Ra2 ND ND ND ND Eotaxin-1 2.267 2.729 0.2670.182 IL-10 0.033** 0.049 0.009 0.008 CD40 2.559 3.243 2.215 2.714 CD40L0.747 0.873 0.382 0.494 ^(a)Statistical analysis for differences betweengroups was performed with one-way ANOVA with Tukey's post-comparisontest of all groups (**p < 0.01)

When animals were compared based on OVA versus PBS challenge, the datasuggest that wildtype mice are more likely to significantly increasegene levels following OVA challenge compared to IL-31 transgenicanimals. Data show that the up-regulation of genes such as IL-4, IL-5,IL-13 and IL-31Ra were more significantly up-regulated in wildtypes OVAsensitized animals compared to PBS sensitized mice than transgenic OVAsensitized compared to transgenic PBS mice. Some genes like Eotaxin andClass II were equally well up-regulated in either the wildtype or IL-31transgenic OVA sensitized animals compared to their PBS controls (Table18).

TABLE 18 mRNA Levels in Total Lung Homogenates from IL-31 Transgenicmice following Sensitization and Challenge with OVA - Comparison of OVAversus PBS ^(a)IL-31 Tg ^(a)Wildtype Gene OVA PBS OVA PBS IL-4 0.0200.001 0.041* 0.001 IL-5 0.188 0.105 0.300* 0.093 IL-13 0.365 0.0010.645** 0.001 IL-31Ra 0.601 0.179 1.023* 0.192 IFNg 0.318 0.110 0.3240.136 TNFa 0.973 0.797 0.654 0.693 MIP-2 0.946 1.302 1.878 0.539 IL-8R3.666 4.205 4.136 3.696 CCL27 0.511*** 0.806 0.528*** 0.814 Cathepsin L34.840* 45.421 43.339** 55.763 Class II 49.180*** 24.050 50.207** 33.128IL-13Ra2 ND ND ND ND Eotaxin-1 2.267** 0.267 2.729** 0.182 IL-10 0.0330.009 0.049** 0.008 CD40 2.559 2.215 3.243 2.714 CD40L 0.747 0.382 0.8730.494 ^(a)Statistical analysis for differences between groups wasperformed with one-way ANOVA with Tukey's post-comparison test of allgroups (***P < 0.001, **p < 0.01, *p < 0.05)

Analysis of mRNA from BAL cell infiltrates comparing IL-31 transgenic towildtype controls under conditions of either OVA or PBS intranasalchallenge suggests (i) three genes tested, TNFa, IL-8R and Class II,were expressed at significantly different levels in IL-31 transgenicscompared to wildtypes in the absence of a antigen-specific challenge.TNFa and Class II were both lower in transgenics versus wildtypes whereas IL-8R was expressed at higher levels. (ii) Following OVA intranasalchallenge wildtype control mice up-regulated the expression of IL-4,IL-13, IL-8R, Class II, Cathepsin L, Eotaxin, IL-10 and CD40significantly more than the IL-31 transgenic mice. (iii) IL-31transgenic mice up-regulated CD40L following OVA challenge significantlymore than wildtype littermate controls (Table 19).

TABLE 19 mRNA Levels in BAL mRNA from IL-31 Transgenic and Wildtype micefollowing Sensitization and Challenge with OVA - Comparison of Wildtypeversus IL-31 Transgenic ^(a)OVA ^(a)PBS challenge Gene IL-31 Tg WildtypeIL-31 Tg Wildtype IL-4 0.011*** 0.030 ND ND IL-5 0.071 0.102 0.000 0.001IL-13 0.028*** 0.044 ND ND IL-31Ra 0.480 0.520 0.010 0.017 IFNg 0.3950.346 0.025 0.026 TNFa 0.522 0.451 1.924*** 3.316 MIP-2 0.991 1.0314.960 5.278 IL-8R 0.736*** 1.541 0.767*** 0.350 CCL27 0.176 0.168 0.1650.200 Cathepsin L 42.517* 55.496 16.574 24.384  Class II 23.420***29.767 4.733** 9.228 IL-13Ra2 ND ND ND ND Eotaxin-1 0.001*** 0.005 ND NDIL-10 0.050* 0.066 0.008 0.003 CD40 0.229** 0.265 0.033 0.038 CD40L0.707** 0.538 0.052 0.075 ^(a)Statistical analysis for differencesbetween groups was performed with one-way ANOVA with Tukey'spost-comparison test of all groups (***p < 0.001, **p < 0.01, *p < 0.05)

Data in Table 20 clearly show that OVA intranasal challenge induces anumber of genes in both IL-31 transgenic and wildtype OVA treated micecompared to PBS challenge groups. However, two genes, IL-8R and Eotaxinwere not up-regulated in IL-31 transgenic OVA challenged mice comparedto their PBS controls, whereas both genes were up-regulated in thewildtype mice challenged with OVA, compared to PBS controls (Table 20).

TABLE 20 mRNA Levels in BAL mRNA from IL-31 Transgenic mice followingSensitization and Challenge with OVA - Comparison of OVA versus PBS^(a)IL-31 Transgenic ^(a)Wildtype Gene OVA PBS OVA PBS IL-4 0.011*** ND0.030*** ND IL-5 0.071*** 0.000 0.102*** 0.001 IL-13 0.028*** ND0.044*** ND IL-31Ra 0.480*** 0.010 0.520*** 0.017 IFNg 0.395*** 0.0250.346*** 0.026 TNFa 0.522*** 1.924 0.451*** 3.316 MIP-2 0.991*** 4.9601.031*** 5.278 IL-8R 0.736 0.767 1.541*** 0.350 CCL27 0.176 0.165 0.1680.200 Cathepsin L 42.517*** 16.574  55.496*** 24.384  Class II 23.420***4.733 29.767*** 9.228 IL-13Ra2 ND ND ND ND Eotaxin-1 0.001 ND 0.005***ND IL-10 0.050*** 0.008 0.066*** 0.003 CD40 0.229*** 0.033 0.265***0.038 CD40L 0.707*** 0.052 0.538*** 0.075 ^(a)Statistical analysis fordifferences between groups was performed with one-way ANOVA with Tukey'spost-comparison test of all groups (***p < 0.001, **p < 0.01, *p < 0.05)

3.2.4.3.2 BAL Cell Differentials

BAL cell infiltrates were collected and analyzed for cellular content.Although there was a trend towards decreased eosinophil numbers in thelung cell infiltrates of IL-31 Tg animals compared to wildtype controllittermates, the data did not reach statistical significance due tovariability within the test groups.

3.2.4.3.3 Serum OVA-Specific and Total IgE

Analysis of total IgE in the serum of IL-31 transgenic animals followingOVA-sensitization showed significant decreases in the production oftotal IgE in the circulation of IL-31 transgenic animals compared tolittermate wildtype controls (p=0.048, unpaired t test), however therewere no statistical differences in the level of OVA-specific IgEdetectable in the serum.

3.2.4.4 Summary

Given that IL-31 delivery decreases pulmonary inflammation followingallergen sensitization and challenge, we hypothesized that IL-31transgenic animals would also be less susceptible to allergen inducedasthma. Indeed IL-31 transgenics appeared to develop less airwayhyper-responsiveness to OVA sensitization and challenge when airwayresponsiveness was measured by whole body plethysmography. We found thatIL-31 transgenics appeared to be more resistant to the up-regulation ofTh-2-type genes following OVA sensitization and challenge compared tolittermate controls. Moreover, there was a trend towards decreasednumbers of eosinophils in the lungs OVA-challenged IL-31 transgenicsversus wildtype controls. These data are consistent with ourobservations that IL-31 over-expression down-regulates pulmonaryinflammation.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of inhibiting, minimizing, reducing, or neutralizing chronicobstructive pulmonary disease (COPD) in a mammal, comprisingadministering an Interleukin-31 (IL-31) agonist to the mammal whereinpulmonary inflammation is reduced and wherein the IL-31 agonist isadministered during sensitization or challenge and wherein the IL-31agonist comprises amino acid residues 27 to 164 of SEQ ID NO:
 2. 2. Themethod of claim 1, wherein production of proinflammatory cytokines inthe lung and BAL fluid is inhibited, minimized, or neutralized.
 3. Themethod of claim 2, wherein the proinflammatory cytokines are IL-5 orIL-13.
 4. The method of claim 2, wherein the proinflammatory cytokinesare IL-5 and IL-13.
 5. A method of inhibiting, minimizing, reducing, orneutralizing chronic obstructive pulmonary disease (COPD) in a mammal,comprising administering an Interleukin-31 (IL-31) agonist to the mammalwherein pulmonary inflammation is reduced and wherein the IL-31 agonistis administered during sensitization or challenge and wherein the IL-31agonist has at least 95% sequence identity to the polypeptide comprisingthe amino acid sequence of SEQ ID NO:2 from residue 27 to
 164. 6. Themethod of claim 5, wherein the IL-31 agonist is produced in mammaliancells.
 7. The method of claim 5, wherein the IL-31 agonist is producedin E. coli.